Host-pathogen interactions of secreted and surface Staphylococcus aureus factors

Staphylococcus aureus is an opportunistic bacterial pathogen that can infect humans and other species. It utilizes an arsenal of virulence factors to cause disease, including secreted and cell wall anchored factors. Secreted toxins attack host cells, and pore-forming toxins destroy target cells by causing cell lysis. S. aureus uses cell-surface adhesins to attach to host molecules thereby facilitating host colonization. The Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMMs) are a family of cell-wall anchored proteins that target molecules like fibronectin and fibrinogen. The Serine-aspartate repeat (Sdr) proteins are a subset of staphylococcal MSCRAMMs that share similar domain organization. Interestingly, the amino-terminus, is composed of three immunoglobulin-folded subdomains (N1, N2, and N3) that contain ligand-binding activity. Clumping factors A and B (ClfA and ClfB) and SdrG are Sdr proteins that bind to fibrinogen (Fg), a large, plasma glycoprotein that is activated during the clotting cascade to form fibrin. In addition to recognizing fibrinogen, ClfA and ClfB can bind to other host ligands. Analysis of S. aureus strains that cause osteomyelitis led to the discovery of the bone-sialoprotein-binding protein (Bbp), an Sdr protein. Because several MSCRAMMs target more than one molecule, I hypothesized that Bbp may recognize other host proteins. A ligand screen revealed that the recombinant construct BbpN2N3 specifically recognizes human Fg. Surface plasmon resonance was used to determine the affinity of BbpN2N3 for Fg, and a dissociation constant of 540 nM was determined. Binding experiments performed with recombinant Fg chains were used to map the binding of BbpN2N3 to the Fg Aalpha chain. Additionally, Bbp expressed on the surface of Lactococcus lactis and S. aureus Newman bald mediated attachment of these bacteria to Fg Aalpha. To further characterize the interaction between the two proteins, isothermal titration calorimetry and inhibition assays were conducted with synthetic Fg Aalpha peptides. To determine the physiological implications of Bbp binding to Fg, the effect of Bbp on fibrinogen clotting was studied. Results show that Bbp binding to Fg inhibits the formation of fibrin. The consequences of this interaction are currently under investigation. Together, these data demonstrate that human Fg is a novel ligand for Bbp. This study indicates that the MSCRAMM Bbp may aid in staphylococcal attachment by targeting both an extracellular matrix and a blood plasma protein. The implications of these novel findings are discussed.

Influence of blood contamination on bond strength of a self-etching system.

OBJECTIVES: To detect the influence of blood contamination (BC) on the bond strength (BS) of a self-etching bonding system (SES) to enamel and dentine. METHODS: 25 human molars were longitudinally sectioned on the mesio-distal axis in order to obtain 50 specimens, which were embedded in acrylic resin. At first, the specimens were ground to expose a flat surface of enamel, and a bond strength test was performed. Afterwards, the samples were ground again in order to obtain a flat surface of dentine. Ten groups (total: n=100) were assigned according to substrate (enamel and dentine), step in the bonding sequence when contamination occurred (before the acidic primer and after the bonding resin), and contamination treatment (dry or rinse and dry procedure). Fresh human blood was introduced either before or after SES application (Clearfil SE Bond) and treated with air drying, or by rinsing and drying following application. Composite resin (Filtek Z-250,3M ESPE) was applied as inverted, truncated cured cones that were debonded in tension. RESULTS: The mean tensile BS values (MPa) for enamel/dentine were 19.4/23.0 and 17.1/10.0 for rinse-and-dry treatment (contamination before and after SES, respectively); while the measurements for the dry treatment, 16.2/23.3 and 0.0/0.0 contamination before and after SES, respectively. CONCLUSIONS: It was determined that blood contamination impaired adhesion to enamel and dentine when it occurred after bond light curing. Among the tested contamination treatments, the rinse-and-dry treatment produced the highest bond strength with BC after SES application, but it was not sufficient to recover the BS in the contamination-free group.

Polybrominated diphenyl ethers: Potential human exposure through household dust and dryer lint

This MPH thesis consists of (1) literature review of the relatively new synthetic persistent organic pollutants (POP), polybrominated diphenyl ethers (PBDEs), a type of flame retardant posing a potential public health hazard, (2) Presentation of data on PBDE levels in dryer lint from Dallas, TX and Hamburg, Germany. ^ PBDEs are used as additive fire retardants in plastics, polyurethane foam and electronic equipment to reduce flammability and thus save life and property. PBDEs have been widely used beginning in the 1970s. They resemble polychlorinated biphenyls (PCBs) in structure and toxicity. PBDEs are found in environmental sediments, sludges, and wildlife and even in human blood, milk and tissues. ^ PBDEs, due to their lipophilicity, accumulate in fat and other tissues and biomagnify up the food chain, with increasing concentrations. Animal studies have suggested potential health effects including thyroid disruption, permanent learning and memory impairment, fetal malformations, developmental neurotoxicity and, at high doses, possibly cancer. ^ PBDE levels are increasing in blood and breast milk in North America, but PBDEs intake unlike PCBs appears to be not primarily through food; food PBDE levels in the U.S. are not markedly higher than in Europe yet U.S. human blood and milk levels are much higher. For this reason various exposure pathways including PBDE contaminated dust and air have been studied to better characterize routes of PBDE intake into humans. ^ The scientific literature on PBDE levels in household dust reports higher PBDE concentration in dust than that found in dryer lint; levels in the U.S are elevated compared to other countries with congeners such as BDE 47, 99, 100 and 209 predominating. The United Kingdom has elevated BDE 209 due to high usage of Deca commercial mixture. These studies suggest that indoor PBDE contamination through household dust could be a potential source of PBDE exposure and body burden especially in young children. ^ PBDE levels in dryer lint from U.S ranged from 321 to 3073 ng/g (Mean: 1138 ng/g, Median: 803 ng/g) and from Germany were from 330 to 2069 ng/g (Median: 71ng/g, Mean: 361 ng/g). High median levels in U.S samples indicate contamination of lint with PBDEs although the source of the PBDEs in lint may be from dryer electrical components or air deposition onto clothes, lint may be one source of PBDE exposure to humans. ^

Measurement of the vascular input function in mice for DCE-MRI

DCE-MRI is an important technique in the study of small animal cancer models because its sensitivity to vascular changes opens the possibility of quantitative assessment of early therapeutic response. However, extraction of physiologically descriptive parameters from DCE-MRI data relies upon measurement of the vascular input function (VIF), which represents the contrast agent concentration time course in the blood plasma. This is difficult in small animal models due to artifacts associated with partial volume, inflow enhancement, and the limited temporal resolution achievable with MR imaging. In this work, the development of a suite of techniques for high temporal resolution, artifact resistant measurement of the VIF in mice is described. One obstacle in VIF measurement is inflow enhancement, which decreases the sensitivity of the MR signal to the presence of contrast agent. Because the traditional techniques used to suppress inflow enhancement degrade the achievable spatiotemporal resolution of the pulse sequence, improvements can be achieved by reducing the time required for the suppression. Thus, a novel RF pulse which provides spatial presaturation contemporaneously with the RF excitation was implemented and evaluated. This maximizes the achievable temporal resolution by removing the additional RF and gradient pulses typically required for suppression of inflow enhancement. A second challenge is achieving the temporal resolution required for accurate characterization of the VIF, which exceeds what can be achieved with conventional imaging techniques while maintaining adequate spatial resolution and tumor coverage. Thus, an anatomically constrained reconstruction strategy was developed that allows for sampling of the VIF at extremely high acceleration factors, permitting capture of the initial pass of the contrast agent in mice. Simulation, phantom, and in vivo validation of all components were performed. Finally, the two components were used to perform VIF measurement in the murine heart. An in vivo study of the VIF reproducibility was performed, and an improvement in the measured injection-to-injection variation was observed. This will lead to improvements in the reliability of quantitative DCE-MRI measurements and increase their sensitivity.

Polybrominated diphenyl ether (PBDE) exposure in North Americans: Cause for concern?

Polybrominated diphenyl ethers (PBDEs) are brominated flame retardants (BFRs) that have been widely produced and used as flame retardants since the 1970’s in many consumer products such as carpet and drape linings, plastics used in electronics, computer and television casings and polyurethane foam used in chairs, sofas and mattresses. PBDEs are persistent organic pollutants (POPs), which, by definition, are toxic in nature, persistent in the environment and accumulative in living organisms. Animal studies have found PBDEs to cause health defects such as fetal malformations, delayed onset of puberty, decreased sperm count, behavioral changes, permanent learning and memory impairment, endocrine disruption, as well as cancer at high doses. Recent research involving humans reported that elevated breast milk PBDEs levels in their mothers are associated with cryptorchidism (absence of one or both testes from the scrotum) in newborn boys and adverse birth outcomes as well as elevated serum PBDE levels in mothers are associated with low sperm count in young men. There are three commonly manufactured PBDE commercial mixtures: Penta-, Octa-, and Deca-BDEs. Two of them (Octa- and Penta-BDEs) have been banned by the European Union and are being voluntarily phased out in the United States. However, Deca continues to be manufactured, used, and imported in the United States. This MPH thesis consists of a literature review of peer reviewed scientific articles concerned with PBDEs in the environment and in humans, as well as a discussion concerning different routes of exposure to PBDEs and their blood, milk and tissue levels as surrogates for body burdens in North Americans and in people from other countries. Results of this literature review shows PBDE levels in human blood, milk and tissues are higher in North Americans than people from other countries worldwide. To date, the highest level of PBDEs was found in a toddler’s blood in a California study. Despite the fact that PBDEs are associated with adverse health effects, and highest levels of PBDEs in North Americans, Deca-BDE is still manufactured, used and imported in the United States. There is an urgent need of new federal regulatory policy to ban completely the production, importation and use of all commercial mixtures of PBDEs.^

THE OCCURRENCE OF GROUP G STREPTOCOCCUS IN THE DOMESTIC CAT: COMPARISON OF THE PHYSIOLOGICAL PROPERTIES OF GROUP G STREPTOCOCCUS ISOLATED FROM HUMANS AND CATS

The occurrence of group G streptococci in cats and evaluation of the recovered organisms as potential human pathogens was investigated. Throat swabs were obtained from 89 cats (47 males and 42 females) and vaginal swabs from 39 female cats. Eighty-three of the examined cats were housed in individual cages at a University Animal Care Facility. Six cats, 2 mature males, 2 mature females and 2 young females were family pets in a rural area. Beta-hemolytic streptococci were recovered from 33 (37%) of the 89 cat throats cultured, and 27 (30.3%) were identified as group G. More males (34%) than females (24%) had throat cultures positive for group G. From the 39 vaginal cultures examined, 24 (61.5%) contained beta-hemolytic streptococci and 23 (58.9%) were identified as group G streptococci. Streptococci were not recovered from the vaginal cultures of the 5 females under 6 months of age.^ Thirty one group G streptococci isolated from cats were compared with 37 isolates of group G obtained from humans (health status or site of origin unknown). More group G cat isolates (81%) produced deoxyribonuclease (DNase) than did the human isolates (36%). The proportion of cat throat and vaginal isolates producing DNase was the same. Production of nicotinamide adenine dinucleotide glycohydrolase (NADase) by group G isolates of human origin was 70%, cat throat isolates 53% and cat vaginal isolates 37%. The Serum Opacity Factor was present in 73% of the cat throat isolates of group G, 43.7% of the cat vaginal isolates and 58.6% of the human isolates. Possession of an anti-phagocytic factor (M protein like substance) demonstrated by the ability to multiply in fresh human blood was greater in the group G from cat throats (46.7%) than from cat vagina (37.5%) or from the human isolates (13.5%). Many of the biochemical characteristics of the group G streptococci of cat origin were more similar to the biochemical characteristics of group A streptococci, than to the characteristics of group G of human origin. The group G streptococci, found in a large number of cats, could be potential human pathogens, as their physiological and biological characteristics are very similar to those of group A, a known human pathogen. ^

BLyS/BAFF-R signaling pathway in human aggressive non Hodgkin's lymphoma B cell and normal peripheral blood B lymphocytes

B-lymphocyte stimulator (BLyS also called BAFF), is a potent cell survival factor expressed in many hematopoietic cells. BLyS levels are elevated in the serum of non-Hodgkin lymphoma (NHL) patients, and have been reported to be associated with disease progression, and prognosis. To understand the mechanisms involved in BLyS gene expression and regulation, we examined expression, function, and regulation of the BLyS gene in B cell non-Hodgkin's lymphoma (NHL-B) cells. BLyS is constitutively expressed in aggressive NHL-B cells including large B cell lymphoma (LBCL) and mantle cell lymphoma (MCL) contributing to survival and proliferation of malignant B cells. Two important transcription factors, NF-κB and NFAT, were found to be involved in regulating BLyS expression through at least one NF-κB and two NFAT binding sites in the BLyS promoter. Further study indicates that the constitutive activation of NF-κB and BLyS in NHL-B cells forms a positive feedback loop contributing to cell survival and proliferation. In order to further investigate BLyS signaling pathway, we studied the function of BAFF-R, a major BLyS receptor, on B cells survival and proliferation. Initial study revealed that BAFF-R was also found in the nucleus, in addition to its presence on plasma membrane of B cells. Nuclear presentation of BAFF-R can be increased by anti-IgM and soluble BLyS treatment in normal peripheral B lymphocytes. Inhibition of BLyS expression decreases nuclear BAFF-R level in LBCL cells. Furthermore, we showed that BAFF-R translocated to nucleus through the classic karyopherin pathway. A candidate nuclear localization sequence (NLS) was identified in the BAFF-R protein sequence and mutation of this putative NLS can block BAFF-R entering nucleus and LBCL cell proliferation. Further study showed that BAFF-R co-localized with NF-κB family member, c-rel in the nucleus. We also found BAFF-R mediated transcriptional activity, which could be increased by c-rel. We also found that nuclear BAFF-R could bind to the NF-κB binding site on the promoters of NF-κB target genes such as BLyS, CD154, Bcl-xL, Bfl-1/A1 and IL-8. These findings indicate that BAFF-R may also promote survival and proliferation of normal B cells and NHL-B cells by directly functioning as a transcriptional co-factor with NF-κB family member. ^

Tracking of plasma lipids and lipoproteins, within-person and year-to-year variability in plasma cholesterol levels among participants from age 8 to 18 in Project HeartBeat!

Coronary heart disease remains the leading cause of death in the United States and increased blood cholesterol level has been found to be a major risk factor with roots in childhood. Tracking of cholesterol, i.e., the tendency to maintain a particular cholesterol level relative to the rest of the population, and variability in blood lipid levels with increase in age have implications for cholesterol screening and assessment of lipid levels in children for possible prevention of further rise to prevent adulthood heart disease. In this study the pattern of change in plasma lipids, over time, and their tracking were investigated. Also, within-person variance and retest reliability defined as the square root of within-person variance for plasma total cholesterol, HDL-cholesterol, LDL-cholesterol, and triglycerides and their relation to age, sex and body mass index among participants from age 8 to 18 years were investigated. ^ In Project HeartBeat!, 678 healthy children aged 8, 11 and 14 years at baseline were enrolled and examined at 4-monthly intervals for up to 4 years. We examined the relationship between repeated observations by Pearson's correlations. Age- and sex-specific quintiles were calculated and the probability of participants to remain in the uppermost quintile of their respective distribution was evaluated with life table methods. Plasma total cholesterol, HDL-C and LDL-C at baseline were strongly and significantly correlated with measurements at subsequent visits across the sex and age groups. Plasma triglyceride at baseline was also significantly correlated with subsequent measurements but less strongly than was the case for other plasma lipids. The probability to remain in the upper quintile was also high (60 to 70%) for plasma total cholesterol, HDL-C and LDL-C. ^ We used a mixed longitudinal, or synthetic cohort design with continuous observations from age 8 to 18 years to estimate within person variance of plasma total cholesterol, HDL-C, LDL-C and triglycerides. A total of 5809 measurements were available for both cholesterol and triglycerides. A multilevel linear model was used. Within-person variance among repeated measures over up to four years of follow-up was estimated for total cholesterol, HDL-C, LDL-C and triglycerides separately. The relationship of within-person and inter-individual variance with age, sex, and body mass index was evaluated. Likelihood ratio tests were conducted by calculating the deviation of −2log (likelihood) within the basic model and alternative models. The square root of within-person variance provided the retest reliability (within person standard deviation) for plasma total cholesterol, HDL-C, LDL-C and triglycerides. We found 13.6 percent retest reliability for plasma cholesterol, 6.1 percent for HDL-cholesterol, 11.9 percent for LDL-cholesterol and 32.4 percent for triglycerides. Retest reliability of plasma lipids was significantly related with age and body mass index. It increased with increase in body mass index and age. These findings have implications for screening guidelines, as participants in the uppermost quintile tended to maintain their status in each of the age groups during a four-year follow-up. The magnitude of within-person variability of plasma lipids influences the ability to classify children into risk categories recommended by the National Cholesterol Education Program. ^

The roles of costimulatory molecules in the cooperative effects of combination epinephrine and dexamethasone on type-1/type-2 cytokine production in tetanus-toxoid specific stimulated human peripheral blood mononuclear cells

A growing number of studies show strong associations between stress and altered immune function. In vivo studies of chronic and acute stress have demonstrated that cognitive stressors are strongly correlated with high circulating levels of catecholamines (CT) and corticosteroids (CS) that are associated with changes in type-1/type-2 cytokine expression. Although individual pharmacologic doses of CS and CT can inhibit the expression of T-helper 1 (Th1, type-1 like) and promote the production of T-helper 2 (Th2, type-2 like) cytokines in antigen-specific and mitogen stimulated human leukocyte cultures in vitro, little attention has been focused on the effects of combination physiologic-stress doses of CT and CS that may be more physiologically relevant. In addition, both in-vivo and in-vitro studies suggest that the differential expression of the B7 family of costimulatory molecules CD80 and CD86 may promote the expression of type-1 or type-2 cytokines, respectively. Furthermore, corticosteroids can influence the expression of β2-adrenergic receptors in various human tissues. We therefore investigated the combined effects of physiologic-stress doses of in vitro CT and CS upon the type-1/type-2 cytokine balance and expression of B7 costimulatory molecules of human peripheral blood mononuclear cells (PBMC) as a model to study the immunomodulatory effects of physiologic stress. Results demonstrated a significant decrease in type-1 cytokine expression and a significant increase in type-2 cytokine production in our CS+CT incubated cultures when compared to either CT or CS agents alone. In addition, we demonstrated the differential expression of CD80/CD86 in favor of CD86 at the cellular and population level as determined by flow cytometry in lipopolysaccharide stimulated human Monocytes. Furthermore, we developed flow cytometry based assays to detect total β2AR in human CD4+ T-lymphocytes that demonstrated decreased expression of β2AR in mitogen stimulated CD4+ T-lymphocytes in the presence of physiologic stress levels of CS and CT as single in vitro agents, however, when both CS and CT were combined, significantly higher expression of β2AR was observed. In summary, our in vitro data suggest that both CS and CT work cooperatively to shift immunity towards type-2 responses. ^

Genetic studies of the human plasma orosomucoid (alpha-1 acid glycoprotein)

Orosomucoid (ORM) or alpha-1 acid glycoprotein is an acute phase protein of human plasma whose function is suggested to be the competitive inhibition of cellular recognition by infective agents. Isoelectric focusing (IEF) and immunoblotting have been combined and optimum conditions have been determined for reliable classification of different ORM phenotypes. Addition of 6 M urea in an IEF gel revealed additional microheterogeneity in the ORM system which has not been previously reported. 1,667 individuals from different native ethnic groups of North and South America, Africa and New Guinea have been screened to determine the distribution of ORM alleles. Two common alleles, ORM1*1 and ORM1*2 have been observed and their frequencies were determined. Genetically independent variation consistent with expression of the ORM2 locus was observed in American and African blacks but was not observed in other sampled populations. The population allele frequencies for this new locus were 0.958, 0.025, 0.006, 0.011, for alleles ORM2*1, ORM2*2, ORM2*3, ORM2*4, respectively. Family studies confirm the autosomal codominant inheritance of the phenotypes observed at both ORM loci. ^

EVIDENCE OF HUMAN ENDOGENOUS RETROVIRUS K INVOLVEMENT IN HUMAN CANCER

Many lines of clinical and experimental evidence indicate a viral role in carcinogenesis (1-6). Our access to patient plasma, serum, and tissue samples from invasive breast cancer (N=19), ductal carcinoma in situ (N=13), malignant ovarian cancer (N=12), and benign ovarian tumors (N=9), via IRB-approved and informed consent protocols through M.D. Anderson Cancer Center, as well as normal donor plasmas purchased from Gulf Coast Regional Blood Center (N=6), has allowed us to survey primary patient blood and tissue samples, healthy donor blood from the general population, as well as commercially available human cell lines for the presence of human endogenous retrovirus K (HERV-K) Env viral RNA (vRNA), protein, and viral particles. We hypothesize that HERV-K proteins are tumor-associated antigens and as such can be profiled and targeted in patients for diagnostic and therapeutic purposes. To test this hypothesis, we employed isopycnic ultracentrifugation, a microplate-based reverse transcriptase enzyme activity assay, reverse transcription – polymerase chain reaction (RT-PCR), cDNA sequencing, SDS-PAGE and western blotting, immunofluorescent staining, confocal microscopy, and transmission electron microscopy to evaluate v HERV-K activation in cancer. Data from large numbers of patients tested by reverse transcriptase activity assay were analyzed statistically by t-test to determine the potential use of this assay as a diagnostic tool for cancer. Significant reverse transcriptase enzyme activity was detected in 75% of ovarian cancer patients, 53.8% of ductal carcinoma in situ patient, and 42.1% of invasive breast cancer patient samples. Only 11.1% of benign ovarian patient and 16.7% of normal donor samples tested positive. HERV-K Env vRNA, or Env SU were detected in the majority of cancer types screened, as demonstrated by the results shown herein, and were largely absent in normal controls. These findings support our hypothesis that the presence of HERV-K in patient blood circulation is an indicator of cancer or pre-malignancy in vivo, that the presence of HERV-K Env on tumor cell surfaces is indicative of malignant phenotype, and that HERV-K Env is a tumor-associated antigen useful not only as a diagnostic screening tool to predict patient disease status, but also as an exploitable therapeutic target for various novel antibody-based immunotherapies.