Safety and Biodistribution Profile of Poly(styrenyl acetal trehalose) and Its Granulocyte Colony Stimulating Factor Conjugate.

Poly(styrenyl acetal trehalose) (pSAT), composed of trehalose side chains linked to a polystyrene backbone via acetals, stabilizes a variety of proteins and enzymes against fluctuations in temperature. A promising application of pSAT is conjugation of the polymer to therapeutic proteins to reduce renal clearance. To explore this possibility, the safety of the polymer was first studied. Investigation of acute toxicity of pSAT in mice showed that there were no adverse effects of the polymer at a high (10 mg/kg) concentration. The immune response (antipolymer antibody and cytokine production) in mice was also studied. No significant antipolymer IgG was detected for pSAT, and only a transient and low level of IgM was elicited. pSAT was also safe in terms of cytokine response. The polymer was then conjugated to a granulocyte colony stimulating factor (GCSF), a therapeutic protein that is approved by the Federal Drug Administration, in order to study the biodistribution of a pSAT conjugate. A site-selective, two-step synthesis approach was developed for efficient conjugate preparation for the biodistribution study resulting in 90% conjugation efficiency. The organ distribution of GCSF-pSAT was measured by positron emission tomography and compared to controls GCSF and GCSF-poly(ethylene glycol), which confirmed that the trehalose polymer conjugate improved the in vivo half-life of the protein by reducing renal clearance. These findings suggest that trehalose styrenyl polymers are promising for use in therapeutic protein-polymer conjugates for reduced renal clearance of the biomolecule.

Quality Control Practices for Chemistry and Immunochemistry in a Cohort of 21 Large Academic Medical Centers.

OBJECTIVES: In the United States, minimum standards for quality control (QC) are specified in federal law under the Clinical Laboratory Improvement Amendment and its revisions. Beyond meeting this required standard, laboratories have flexibility to determine their overall QC program. METHODS: We surveyed chemistry and immunochemistry QC procedures at 21 clinical laboratories within leading academic medical centers to assess if standardized QC practices exist for chemistry and immunochemistry testing. RESULTS: We observed significant variation and unexpected similarities in practice across laboratories, including QC frequency, cutoffs, number of levels analyzed, and other features. CONCLUSIONS: This variation in practice indicates an opportunity exists to establish an evidence-based approach to QC that can be generalized across institutions.

Elimination of Mycoplasma contamination in Chlamydia stocks as a result of in vivo passage or plaque isolation.

OBJECTIVE: This study aims to eliminate Mycoplasma spp. contamination from laboratory stocks of Chlamydia spp. by in vivo passage or by plaque assay. RESULTS: We have described two methods of eliminating Mycoplasma contamination from Chlamydia laboratory stocks. We conclude that Mycoplasma species commonly contaminating chlamydial stocks do not survive passage in mice. Chlamydia may also be derived Mycoplasma-free by plaque assay.

Integrating the social determinants of health into two interprofessional courses: Findings from a pilot study.

Five colleges and universities in Upstate New York, United States, created the 'Route-90 Collaborative' to support faculty implementing the Institute of Medicine's (IOM) Framework for Educating Health Professionals to Address the Social Determinants of Health. The two courses described herein used a flipped classroom approach in which students from 14 different nations were responsible for facilitating individual classes. This descriptive study used an educational intervention in two interprofessional courses - reproductive health and global health - based on the IOM Framework into two courses. The evaluation used quantitative and open-ended text response data from students. Course evaluations indicated the students found the courses helped them to learn more about health issues and service delivery in various countries, expand their knowledge base on sociocultural and ecological influences on health care, and broaden their perspectives on various health topics so they will be able to provide higher quality healthcare. Although this is the first effort of our Collaborative to implement the Framework, given the student feedback, we believe implementing the Framework in various courses has the potential to enhance healthcare service delivery and reduce the negative impact of social determinants of health.

Potentially Preventable Hospitalizations and the Burden of Healthcare-Associated Infections.

BACKGROUND: An estimated 4% of hospital admissions acquired healthcare-associated infections (HAIs) and accounted for $9.8 (USD) billion in direct cost during 2011. In 2010, nearly 140 000 of the 3.5 million potentially preventable hospitalizations (PPHs) may have acquired an HAI. There is a knowledge gap regarding the co-occurrence of these events. AIMS: To estimate the period occurrences and likelihood of acquiring an HAI for the PPH population. METHODS: Retrospective, cross-sectional study using logistic regression analysis of 2011 Texas Inpatient Discharge Public Use Data File including 2.6 million admissions from 576 acute care hospitals. Agency for Healthcare Research and Quality Prevention Quality Indicator software identified PPH, and existing administrative data identification methodologies were refined for Clostridium difficile infection, central line-associated bloodstream infection, catheter-associated urinary tract infection, and ventilator-associated pneumonia. Odds of acquiring HAIs when admitted with PPH were adjusted for demographic, health status, hospital, and community characteristics. FINDINGS: We identified 272 923 PPH, 14 219 HAI, and 986 admissions with PPH and HAI. Odds of acquiring an HAI for diabetic patients admitted for lower extremity amputation demonstrated significantly increased odds ratio of 2.9 (95% confidence interval: 2.16-3.91) for Clostridium difficile infection. Other PPH patients had lower odds of acquiring HAI compared to non-PPH patients, and results were frequently significant. CONCLUSIONS: Clinical implications include increased risk of HAI among diabetic patients admitted for lower extremity amputation. Methodological implications include identification of rare events for inpatient subpopulations and the need for improved codification of HAIs to improve cost and policy analyses regarding allocation of resources toward clinical improvements.

Identification of leachables observed in the size exclusion chromatograms of a low concentration product stored in prefilled syringes.

Requisite leachables testing of pharmaceutical products is commonly conducted with pre-defined analytical methods on a subset of materials intended to be representative of the marketed product. Throughout product development, leachables may occasionally be detected in other methods not specifically intended for monitoring such impurities. We have identified two leachables, ethyl 4-ethoxybenzoate (E4E) and 2,6-di(t-butyl)-4-hydroxy-4-methyl-2,5-cyclohexadien-1-one (BHT-OH) in a low concentration product stored in prefilled syringes (PFS). The leachables were initially detected by size exclusion chromatography (SEC) as late-eluting impurity peaks. Syringe component extraction studies indicated that the impurities were related to the syringe stoppers. Positive identification of E4E was accomplished by reversed phase liquid chromatography- tandem mass spectrometry (RPLC-MS/MS). Positive identification of BHT-OH required RPLC-solid phase extraction-cryoflow NMR (RPLC-SPE-NMR), as initial RPLC-MS/MS investigations were unsuccessful in elucidating the structure. We focus specifically on the efforts required to identify the leachables, and the fortuitous mixed mode separation mechanism and low concentration nature of the product, which were the main factors contributing to the unlikely detection of the leachables by SEC. We note that our investigations were conducted independently of formal leachables and extractables (L&E) studies and we discuss challenges with designing and conducting such studies in a manner that captures the comprehensive L&E profile of a product.

A Protective Vaccine against Chlamydia Genital Infection Using Vault Nanoparticles without an Added Adjuvant.

Chlamydia trachomatis genital infection is the most common sexually transmitted bacterial disease, causing a significant burden to females due to reproductive dysfunction. Intensive screening and antibiotic treatment are unable to completely prevent female reproductive dysfunction, thus, efforts have become focused on developing a vaccine. A major impediment is identifying a safe and effective adjuvant which induces cluster of differentiation 4 (CD4) cells with attributes capable of halting genital infection and inflammation. Previously, we described a natural nanocapsule called the vault which was engineered to contain major outer membrane protein (MOMP) and was an effective vaccine which significantly reduced early infection and favored development of a cellular immune response in a mouse model. In the current study, we used another chlamydial antigen, a polymorphic membrane protein G-1 (PmpG) peptide, to track antigen-specific cells and evaluate, in depth, the vault vaccine for its protective capacity in the absence of an added adjuvant. We found PmpG-vault immunized mice significantly reduced the genital bacterial burden and histopathologic parameters of inflammation following a C. muridarum challenge. Immunization boosted antigen-specific CD4 cells with a multiple cytokine secretion pattern and reduced the number of inflammatory cells in the genital tract making the vault vaccine platform safe and effective for chlamydial genital infection. We conclude that vaccination with a Chlamydia-vault vaccine boosts antigen-specific immunities that are effective at eradicating infection and preventing reproductive tract inflammation.

Expression of CXCR3 on Adaptive and Innate Immune Cells Contributes Oviduct Pathology throughout Chlamydia muridarum Infection.

CXCR3 is a chemokine receptor expressed on a wide range of leukocytes, and it is involved in leukocyte migration throughout the blood and lymphatics. Specifically, CXCR3 is required for lymphocyte homing to the genital mucosa. When compared to wild type (WT) mice, CXCR3 deficiency (CXCR3-/-) mice infected with Chlamydia muridarum (C. muridarum) did not display impaired clearance and resolution of infection. However, they possessed significantly higher bacterial burden and lower levels of IFN-γ-producing TH1 cells. The knockouts also demonstrated a significant decrease in the level of activated conventional dendritic cells in the GT, ultimately leading to the decrease in activated TH1 cells. In addition, few activated plasmacytoid dendritic cells, which possess an inflammatory phenotype, were found in the lymph node of infected mice. This reduction in pDCs may be responsible for the decrease in neutrophils, which are acute inflammatory cells, in the CXCR3-/- mice. Due to the significantly reduced level of acute inflammation, these mice also possess a decrease in dilation and pathology in the oviduct. This demonstrates that the CXCR3-/- mice possess the ability to clear C. muridarum infections, but they do so without the increased inflammation and pathology in the GT.

Evaluation of 2 Batched Pretreatment Systems for the Measurement of Whole Blood Tacrolimus on the ARCHITECT Immunoassay Analyzer.

BACKGROUND: Measurement of tacrolimus using the ARCHITECT immunoassay analyzer requires a manual extraction step that puts clinical laboratory workers at risk for ergonomic injury. Therefore, we developed 2 batched extraction systems for tacrolimus measurement on the ARCHITECT analyzer and describe their features herein. METHODS: Two batched extraction methods were developed at 2 different laboratories. The batched extraction methods allow processing of at least 20 specimens at a time. We evaluated the analytical performance of those methods and compared them with the United States Food and Drug Administration (FDA)-cleared process for manually extracting individual specimens. RESULTS: Comparing the performance of batched- and individual-extraction methods revealed that both methods had comparable between-day imprecision, high patient-results correlation (R2 values ≥0.9869), equivalent functional sensitivity (0.48 ng/mL), and good linearity between 1 ng per mL and 25 ng per mL. Further, we observed decreased delta check-identified errors using the batched method. CONCLUSION: The 2 developed batched extraction methods for tacrolimus measurement that we describe herein demonstrate excellent performance and can replace individual specimen extraction.

Activation of the NLRP3 inflammasome by vault nanoparticles expressing a chlamydial epitope.

The full potential of vaccines relies on development of effective delivery systems and adjuvants and is critical for development of successful vaccine candidates. We have shown that recombinant vaults engineered to encapsulate microbial epitopes are highly stable structures and are an ideal vaccine vehicle for epitope delivery which does not require the inclusion of an adjuvant. We studied the ability of vaults which were engineered for use as a vaccine containing an immunogenic epitope of Chlamydia trachomatis, polymorphic membrane protein G (PmpG), to be internalized into human monocytes and behave as a "natural adjuvant". We here show that incubation of monocytes with the PmpG-1-vaults activates caspase-1 and stimulates IL-1ß secretion through a process requiring the NLRP3 inflammasome and that cathepsin B and Syk are involved in the inflammasome activation. We also observed that the PmpG-1-vaults are internalized through a pathway that is transiently acidic and leads to destabilization of lysosomes. In addition, immunization of mice with PmpG-1-vaults induced PmpG-1 responsive CD4(+) cells upon re-stimulation with PmpG peptide in vitro, suggesting that vault vaccines can be engineered for specific adaptive immune responses. We conclude that PmpG-1-vault vaccines can stimulate NLRP3 inflammasomes and induce PmpG-specific T cell responses.

HIV cure strategies: how good must they be to improve on current antiretroviral therapy?

BACKGROUND: We examined efficacy, toxicity, relapse, cost, and quality-of-life thresholds of hypothetical HIV cure interventions that would make them cost-effective compared to life-long antiretroviral therapy (ART). METHODS: We used a computer simulation model to assess three HIV cure strategies: Gene Therapy, Chemotherapy, and Stem Cell Transplantation (SCT), each compared to ART. Efficacy and cost parameters were varied widely in sensitivity analysis. Outcomes included quality-adjusted life expectancy, lifetime cost, and cost-effectiveness in dollars/quality-adjusted life year ($/QALY) gained. Strategies were deemed cost-effective with incremental cost-effectiveness ratios <$100,000/QALY. RESULTS: For patients on ART, discounted quality-adjusted life expectancy was 16.4 years and lifetime costs were $591,400. Gene Therapy was cost-effective with efficacy of 10%, relapse rate 0.5%/month, and cost $54,000. Chemotherapy was cost-effective with efficacy of 88%, relapse rate 0.5%/month, and cost $12,400/month for 24 months. At $150,000/procedure, SCT was cost-effective with efficacy of 79% and relapse rate 0.5%/month. Moderate efficacy increases and cost reductions made Gene Therapy cost-saving, but substantial efficacy/cost changes were needed to make Chemotherapy or SCT cost-saving. CONCLUSIONS: Depending on efficacy, relapse rate, and cost, cure strategies could be cost-effective compared to current ART and potentially cost-saving. These results may help provide performance targets for developing cure strategies for HIV.

Monoclonal and polyclonal immunoglobulin interference in a conjugated bilirubin assay.

CONTEXT: Monoclonal proteins can interfere with the conjugated bilirubin assay on the Beckman Coulter AU5400 and AU2700 instruments and produce spurious results. Protein depletion eliminates the interference, suggesting that monoclonal proteins cause the problem. OBJECTIVES: To determine the interference rate with the Beckman Coulter AU5400 and AU2700 conjugated bilirubin assay and to identify the interfering substance. DESIGN: Beckman Coulter bilirubin results from 33,720 samples analyzed during 6 months were evaluated for interference. On a subset of samples, protein G columns were used to specifically remove immunoglobulin G (IgG) to determine the cause of the interference. Another 117 samples containing a (known) monoclonal protein were selected to determine the interference rate. RESULTS: From 26 different patients, 103 samples had conjugated bilirubin results greater than the total bilirubin for a false-positive rate of 0.3%. Removal of IgG from a subset of those samples with IgG monoclonal protein and increased polyclonal IgG eliminated the conjugated bilirubin interference. In separate, selected samples containing monoclonal proteins, the false-positive interference rate was 7.7% (9 of 117) and was greatest when the monoclonal protein concentration was greater than 4 g/dL. Another 11 of the 117 samples (9.4%) with monoclonal proteins exhibited assay interference by producing false-negative results. The overall interference rate, therefore, was 17.1%. CONCLUSION: The false-positive interference rate for the Beckman Coulter conjugated bilirubin assay was 0.3% for routinely analyzed serum/plasma samples. In addition to monoclonal proteins, we found that polyclonal immunoglobulins can also interfere with the conjugated bilirubin assay. The overall interference rate was 17.1% for samples with a monoclonal protein.

Individualizing the WHO HIV and infant feeding guidelines: optimal breastfeeding duration to maximize infant HIV-free survival.

OBJECTIVES: To determine how infant feeding recommendations can maximize HIV-free survival (HFS) among HIV-exposed, uninfected African infants, balancing risks of breast milk-associated HIV infection with setting-specific risks of illness and death associated with replacement feeding. DESIGN: Validated mathematical model of HIV-exposed, uninfected infants, with published data from Africa. METHODS: We projected 24-month HFS using combinations of: maternal CD4, antiretroviral drug availability, and relative risk of mortality among replacement-fed compared to breastfed infants ('RR-RF', range 1.0-6.0). For each combination, we identified the 'optimal' breastfeeding duration (0-24 months) maximizing HFS. We compared HFS under an 'individualized' approach, based on the above parameters, to the WHO 'public health approach' (12 months breastfeeding for all HIV-infected women). RESULTS: Projected HFS was 65-93%. When the value of RR-RF is 1.0, replacement feeding from birth maximized HFS. At a commonly reported RR-RF value (2.0), optimal breastfeeding duration was 3-12 months, depending on maternal CD4 and antiretroviral drug availability. As the value of RR-RF increased, optimal breastfeeding duration increased. Compared to the public health approach, an individualized approach improved absolute HFS by less than 1% if RR-RF value was 2.0-4.0, by 3% if RR-RF value was 1.0 or 6.0, and by greater amounts if access to antiretroviral drugs was limited. CONCLUSION: Tailoring breastfeeding duration to maternal CD4, antiretroviral drug availability, and local replacement feeding safety can optimize HFS among HIV-exposed infants. An individualized approach leads to moderate gains in HFS, but only when mortality risks from replacement feeding are very low or very high, or antiretroviral drug availability is limited. The WHO public health approach is beneficial in most resource-limited settings.

Oral health care for underserved children in the United States.

Dental caries is the most common chronic disease of childhood with approximately 25% of children from low-income families entering kindergarten without ever having seen a dentist ( Larsen, Larsen, Handwerker, Kim, & Rosenthal, 2009 ). Youth, poverty, and race are characteristics of populations susceptible to oral disease (Dye, Xianfen, & Thorton-Evans, 2012). Services delivering oral health care to underserved populations are referred to as dental safety-net clinics. This article explores the impact of the dental safety-net on improving access to oral health care for underserved children in the United States.

CD8⁺CXCR5⁺ T cells regulate pathology in the genital tract.

We have identified a CD8⁺CXCR5⁺ T cell that prevents the development of oviduct dilation following C. muridarum genital infection. Phenotypic studies show that CD8⁺CXCR5⁺ cells express markers of T regulatory cells (FoxP3, CD25, and GITR) but do not express a necessary component of cytotoxic cells (perforin). Cxcr5⁻/⁻ mice have significantly lower numbers of CD8⁺ cells and lack the CD8⁺CXCR5⁺ population while the total number of CD4⁺ cells is equivalent between mouse strains. The transfer of CD8⁺ splenocytes from WT mice reduces the oviduct dilation seen in Cxcr5⁻/⁻ mice following C. muridarum infection. Future studies will investigate the mechanism by which this cell type regulates genital tract pathology.

Interruption of CXCL13-CXCR5 axis increases upper genital tract pathology and activation of NKT cells following chlamydial genital infection.

BACKGROUND: Regulation of immune responses is critical for controlling inflammation and disruption of this process can lead to tissue damage. We reported that CXCL13 was induced in fallopian tube tissue following C. trachomatis infection. Here, we examined the influence of the CXCL13-CXCR5 axis in chlamydial genital infection. METHODOLOGY AND PRINCIPAL FINDINGS: Disruption of the CXCL13-CXCR5 axis by injecting anti-CXCL13 Ab to BALB/c mice or using Cxcr5-/- mice increased chronic inflammation in the upper genital tract (UGT; uterine horns and oviducts) after Chlamydia muridarum genital infection (GT). Further studies in Cxcr5-/- mice showed an elevation in bacterial burden in the GT and increased numbers of neutrophils, activated DCs and activated NKT cells early after infection. After resolution, we noted increased fibrosis and the accumulation of a variety of T cells subsets (CD4-IFNγ, CD4-IL-17, CD4-IL-10 & CD8-TNFα) in the oviducts. NKT cell depletion in vitro reduced IL-17α and various cytokines and chemokines, suggesting that activated NKT cells modulate neutrophils and DCs through cytokine/chemokine secretion. Further, chlamydial glycolipids directly activated two distinct types of NKT cell hybridomas in a cell-free CD1d presentation assay and genital infection of Cd1d-/- mice showed reduced oviduct inflammation compared to WT mice. CXCR5 involvement in pathology was also noted using single-nucleotide polymorphism analysis in C. trachomatis infected women attending a sub-fertility clinic. Women who developed tubal pathology after a C. trachomatis infection had a decrease in the frequency of CXCR5 SNP +10950 T>C (rs3922). CONCLUSIONS/SIGNIFICANCE: These experiments indicate that disruption of the CXCL13-CXCR5 axis permits increased activation of NKT cells by type I and type II glycolipids of Chlamydia muridarum and results in UGT pathology potentially through increased numbers of neutrophils and T cell subsets associated with UGT pathology. In addition, CXCR5 appears to contribute to inter-individual differences in human tubal pathology following C. trachomatis infection.

Isolation of lymphocytes from mouse genital tract mucosa.

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.

Vault nanocapsules as adjuvants favor cell-mediated over antibody-mediated immune responses following immunization of mice.

BACKGROUND: Modifications of adjuvants that induce cell-mediated over antibody-mediated immunity is desired for development of vaccines. Nanocapsules have been found to be viable adjuvants and are amenable to engineering for desired immune responses. We previously showed that natural nanocapsules called vaults can be genetically engineered to elicit Th1 immunity and protection from a mucosal bacterial infection. The purpose of our study was to characterize immunity produced in response to OVA within vault nanoparticles and compare it to another nanocarrier. METHODOLOGY AND PRINCIPAL FINDINGS: We characterized immunity resulting from immunization with the model antigen, ovalbumin (OVA) encased in vault nanocapsules and liposomes. We measured OVA responsive CD8(+) and CD4(+) memory T cell responses, cytokine production and antibody titers in vitro and in vivo. We found that immunization with OVA contain in vaults induced a greater number of anti-OVA CD8(+) memory T cells and production of IFNγ plus CD4(+) memory T cells. Also, modification of the vault body could change the immune response compared to OVA encased in liposomes. CONCLUSIONS/SIGNIFICANCE: These experiments show that vault nanocapsules induced strong anti-OVA CD8(+) and CD4(+) T cell memory responses and modest antibody production, which markedly differed from the immune response induced by liposomes. We also found that the vault nanocapsule could be modified to change antibody isotypes in vivo. Thus it is possible to create a vault nanocapsule vaccine that can result in the unique combination of immunogen-responsive CD8(+) and CD4(+) T cell immunity coupled with an IgG1 response for future development of vault nanocapsule-based vaccines against antigens for human pathogens and cancer.

Objectified body consciousness in relation to recovery from an eating disorder.

In Western society, the feminine body has been positioned as an object to be looked at and sexually gazed upon; thus, females often learn to view themselves as objects to be observed (i.e., objectified body consciousness (OBC)). This study examined the relation between OBC and eating disorder recovery by comparing its components across non-eating disorder controls, fully recovered, partially recovered, and active eating disorder cases. Results revealed that non-eating disorder controls and fully recovered individuals had similarly low levels of two components of OBC, body surveillance and body shame. Partially recovered individuals looked more similar to those with an active eating disorder on these constructs. The third component of OBC, control beliefs, and a conceptually similar construct, weight/shape self-efficacy, did not differ across groups. Results provide support for the importance of measuring aspects of self-objectification, particularly body surveillance and body shame, across the course of an eating disorder.