Identification and characterization of virulence determinants in the Lyme disease spirochete Borrelia burgdorferi

Borrelia burgdorferi is the etiological agent of Lyme disease, the most common tick-borne disease in the United States. Although the most frequently reported symptom is arthritis, patients can also experience severe cardiac, neurologic, and dermatologic abnormalities. The identification of virulence determinants in infectious B. burgdorferi strains has been limited by their slow growth rate, poor transformability, and general lack of genetic tools. The present study demonstrates the use of transposon mutagenesis for the identification of infectivity-related factors in infectious B. burgdorferi, examines the potential role for chemotaxis in mammalian infection, and describes the development of a novel method for the analysis of recombination events at the Ids antigenic variation locus. A pool of Himar1 mutants was isolated using an infectious B. burgdorferi clone and the transposon vector pMarGent. Clones exhibiting reduced infectivity in mice possessed insertions in virulence determinants putatively involved in host survival and dissemination. These results demonstrated the feasibility of extensive transposon mutagenesis studies for the identification of additional infectivity-related factors. mcp-5 mutants were chosen for further study to determine the role of chemotaxis during infection. Animal studies indicated that mcp-5 mutants exhibited a reduced infectivity potential, and suggested a role for mcp-5 during the early stages of infection. An in vitro phenotype for an mcp-5 mutant was not detected. Genetic complementation of an mcp-5 mutant resulted in restoration of Mcp-5 expression in the complemented clone, as demonstrated by western blotting, but the organisms were not infectious in mice. We believe this result is a consequence of differences in expression between genes located on the linear chromosome and genes present on the circular plasmid used for trans-complementation. Overall, this work implicates mcp-5 as an important determinant of mammalian infectivity. Finally, the development of a computer-assisted method for the analysis of recombination events occurring at the B. burgdorferi vls antigenic variation locus has proven highly valuable for the detailed examination of vls gene conversion. The studies described here provide evidence for the importance of chemotaxis during infection in mice and demonstrate advances in both genetic and computational approaches for the further characterization of the Lyme disease spirochete. ^

Attitudes of Parents at risk of inheriting Li-Fraumeni Syndrome towards predictive genetic testing in their minor-aged children.

Li-Fraumeni Syndrome (LFS) is a hereditary cancer syndrome which predisposes individuals to cancer beginning in childhood. These risks are spread across a lifetime, from early childhood to adulthood. Mutations in the p53 tumor suppressor gene are known to cause the majority of cases of LFS. The risk for early onset cancer in individuals with Li-Fraumeni Syndrome is high. Studies have shown that individuals with LFS have a 90% lifetime cancer risk. Children under 18 have up to a 15% chance of cancer development. Effectiveness of cancer screening and management in individuals with Li-Fraumeni Syndrome is unclear. Screening for LFS-associated cancers has not been shown to reduce mortality. Due to the lack of effective screening techniques for childhood cancers, institutions vary with regard to their policies on testing children for LFS. There are currently no national guidelines regarding predictive testing of children who are at risk of inheriting LFS. No studies have looked at parental attitudes towards predictive p53 genetic testing in their children. This was a cross-sectional pilot study aimed at describing these attitudes. We identified individuals whose children were at risk for inheriting p53 genetic mutations. These individuals were provided with surveys which included validated measures addressing attitudes and beliefs towards genetic testing. The questionnaire included qualitative and quantitative measures. Six individuals completed and returned the questionnaire with a response rate of 28.57%. In general, respondents agreed that parents should have the opportunity to obtain p53 genetic testing for their child. Parents vary in regard to their attitudes towards who should be involved in the decision making process and at what time and under what considerations testing should occur. Testing motivations cited most important by respondents included family history, planning for the future and health management. Concern for insurance genetic discrimination was cited as the most important “con” to genetic testing. Although limited by a poor response rate, this study can give health care practitioners insight into testing attitudes and beliefs of families considering pediatric genetic testing.

Dissemination of methicillin-resistant Staphylococcus aureus USA300 sequence type 8 lineage in Latin America.

BACKGROUND: Methicillin-resistant Staphylococus aureus (MRSA) is an important nosocomial and community-associated (CA) pathogen. Recently, a variant of the MRSA USA300 clone emerged and disseminated in South America, causing important clinical problems. METHODS: S. aureus isolates were prospectively collected (2006-2008) from 32 tertiary hospitals in Colombia, Ecuador, Peru, and Venezuela. MRSA isolates were subjected to antimicrobial susceptibility testing and pulsed-field gel electrophoresis and were categorized as health care-associated (HA)-like or CA-like clones on the basis of genotypic characteristics and detection of genes encoding Panton-Valentine leukocidin and staphylococcal cassette chromosome (SCC) mec IV. In addition, multilocus sequence typing of representative isolates of each major CA-MRSA pulsotype was performed, and the presence of USA300-associated toxins and the arcA gene was investigated for all isolates categorized as CA-MRSA. RESULTS: A total of 1570 S. aureus were included; 651 were MRSA (41%)--with the highest rate of MRSA isolation in Peru (62%) and the lowest in Venezuela (26%)--and 71%, 27%, and 2% were classified as HA-like, CA-like, and non-CA/HA-like clones, respectively. Only 9 MRSA isolates were confirmed to have reduced susceptibility to glycopeptides (glycopeptide-intermediate S. aureus phenotype). The most common pulsotype (designated ComA) among the CA-like MRSA strains was found in 96% of isolates, with the majority (81%) having a < or =6-band difference with the USA300-0114 strain. Representative isolates of this clone were sequence type 8; however, unlike the USA300-0114 strain, they harbored a different SCCmec IV subtype and lacked arcA (an indicator of the arginine catabolic mobile element). CONCLUSION: A variant CA-MRSA USA300 clone has become established in South America and, in some countries, is endemic in hospital settings.

Predictors of HIV-1 slow progression among vertically infected children in Botswana

HIV-1 infected children display a highly variable rate of progression to AIDS. Data about reasons underlying the variable progression to AIDS among vertically-infected children is sparse, and the few studies that have examined this important question have almost exclusively been done in the developed world. This is despite the fact that Sub-Saharan Africa is home to over 90% of all HIV infected children around the world.^ The main objective of this study was to examine predictors of HIV-1 slow progression among vertically infected children in Botswana, using a case control design. Cases (slow progressors) and controls (rapid progressors) were drawn from medical records of HIV-1 infected children being followed up for routine care and treatment at the BBCCCOE between February 2003 and February 2011. Univariate and Multivariate Logistic Regression Analyses were performed to identify independent predictors of slow disease progression and control for confounding respectively. ^ The study population comprised of 152 cases and 201 controls with ages ranging from 6 months to 16 years at baseline. Low baseline HIV-1 RNA viral load was the strongest independent predictor of slow progression (adjusted OR = 5.52, 95% CI = 2.75-11.07; P <0.001). Other independent predictors of slow disease progression identified were: lack of history of PMTCT with single dose Nevirapine plus Zidovudine (adjusted OR = 4.45, 95% CI = 1.45-13.69; P = 0.009) and maternal vital status (alive) (adjusted OR = 2.46, 95% CI = 1.51-4.01; P < 0.00 ).^ The results of this study may help clinicians and policy-makers in resource-limited settings to identify, at baseline, which children are at highest risk of rapid progression to AIDS and thus prioritize them for immediate intervention with HAART and other measures that would mitigate disease progression. At the same time HAART may be delayed among children who are at lower risk of disease progression. This would enable the highly affected, yet impoverished, Sub-Saharan African countries to use their scarce resources more efficiently which may in turn ensure that their National Antiretroviral Therapy Programs become more sustainable. Delaying HAART among the low-risk children would also lower the occurrence of adverse drug reactions associated with antiretroviral drugs exposure.^ Keywords. Slow Progressors, Rapid Progressors, HIV-1, Predictors, Children, Vertical Transmission, Sub-Saharan Africa^

CHARACTERIZATION OF THE COUNT RATE PERFORMANCE AND EVALUATION OF THE EFFECTS OF HIGH COUNT RATES ON MODERN GAMMA CAMERAS

CHARACTERIZATION OF THE COUNT RATE PERFORMANCE AND EVALUATION OF THE EFFECTS OF HIGH COUNT RATES ON MODERN GAMMA CAMERAS Michael Stephen Silosky, B.S. Supervisory Professor: S. Cheenu Kappadath, Ph.D. Evaluation of count rate performance (CRP) is an integral component of gamma camera quality assurance and measurement of system dead time (τ) is important for quantitative SPECT. The CRP of three modern gamma cameras was characterized using established methods (Decay and Dual Source) under a variety of experimental conditions. For the Decay method, input count rate was plotted against observed count rate and fit to the paralyzable detector model (PDM) to estimate τ (Rates method). A novel expression for observed counts as a function of measurement time interval was derived and the observed counts were fit to this expression to estimate τ (Counts method). Correlation and Bland-Altman analysis were performed to assess agreement in estimates of τ between methods. The dependencies of τ on energy window definition and incident energy spectrum were characterized. The Dual Source method was also used to estimate τ and its agreement with the Decay method under identical conditions and the effects of total activity and the ratio of source activities were investigated. Additionally, the effects of count rate on several performance metrics were evaluated. The CRP curves for each system agreed with the PDM at low count rates but deviated substantially at high count rates. Estimates of τ for the paralyzable portion of the CRP curves using the Rates and Counts methods were highly correlated (r=0.999) but with a small (~6%) difference. No significant difference was observed between the highly correlated estimates of τ using the Decay or Dual Source methods under identical experimental conditions (r=0.996). Estimates of τ increased as a power-law function with decreasing ratio of counts in the photopeak to the total counts and linearly with decreasing spectral effective energy. Dual Source method estimates of τ varied as a quadratic with the ratio of the single source to combined source activities and linearly with total activity used across a large range. Image uniformity, spatial resolution, and energy resolution degraded linearly with count rate and image distorting effects were observed. Guidelines for CRP testing and a possible method for the correction of count rate losses for clinical images have been proposed.

TURNOVER RATE OF THE NEURONAL CONNEXIN CX36 IN HELA CELLS

Electrical synapses formed of the gap junction protein Cx36 show a great deal of functional plasticity, much dependent on changes in phosphorylation state of the connexin. However, gap junction turnover may also be important for regulating cell-cell communication, and turnover rates of Cx36 have not been studied. Connexins have relatively fast turnover rates, with short half-lives measured to be 1.5 to 3.5 hours in pulse-chase analyses of connexins (Cx26 and Cx43) in tissue culture cells and whole organs. We utilized HaloTag technology to study the turnover rate of Cx36 in transiently transfected HeLa cells. The HaloTag protein forms irreversible covalent bonds with chloroalkane ligands, allowing pulse-chase experiments to be performed very specifically. The HaloTag open reading frame was inserted into an internal site in the C-terminus of Cx36 designed not to disrupt the regulatory phosphorylation sites and not to block the C-terminal PDZ interaction motif. Functional properties of Cx36-Halo were assessed by Neurobiotin tracer coupling, live cell imaging, and immunostaining. For the pulse-chase study, transiently transfected HeLa cells were pulse labeled with Oregon Green (OG) HaloTag ligand and chase labeled at various times with tetramethylrhodamine (TMR) HaloTag ligand. Cx36-Halo formed large junctional plaques at sites of contact between transfected HeLa cells and was also contained in a large number of intracellular vesicles. The Cx36-Halo transfected HeLa cells supported Neurobiotin tracer coupling that was regulated by activation and inhibition of PKA in the same manner as wild-type Cx36 transfected cells. In the pulse-chase study, junctional protein labeled with the pulse ligand (OG) was gradually replaced by newly synthesized Cx36 labeled with the chase ligand (TMR). The half-life for turnover of protein in junctional plaques was 2.8 hours. Treatment of the pulse-labeled cells with Brefeldin A (BFA) prevented the addition of new connexins to junctional plaques, suggesting that the assembly of Cx36 into gap junctions involves the traditional ER-Golgi-TGN-plasma membrane pathway. In conclusion, Cx36-Halo is functional and has a turnover rate in HeLa cells similar to that of other connexins that have been studied. This turnover rate is likely too slow to contribute substantially to short-term changes in coupling of neurons driven by transmitters such as dopamine, which take minutes to achieve. However, turnover may contribute to longer-term changes in coupling.