Characterization of recombinant class I MHC alloantigen action upon the survival of disparate heart allografts

Previous studies have led to the development of allochimeric class I MHC proteins as agents that effectively induce donor-specific transplantation tolerance in a rat system with or without additional immunosuppression. Within the α1-helical region of RT1.Au, an epitope that conferred immunologic tolerance was discovered. Studies presented herein were designed to test our central hypothesis that allochimeric proteins onfer tolerance in a mouse model. To test this hypothesis, portal vein (PV) injection of wild-type H2Kd and H2Dd proteins were produced in a bacterial expression system and found to specifically prolong the survival of BALB/c (H2d) heart allografts in C57BL/10 (H2b) recipients. Although a single PV injection of 50 μg α1–α 3 H2Kd alone was ineffective, 50 μg α1 –α3 alone slightly prolonged BALB/c heart allograft survivals. In contrast, the combination of 25 μg α1–α 3 H2Kd and 25 μg α1–α 3 H2Dd proteins prolonged BALB/c graft survivals to 20.2 ± 6.4 days (p < 0.004). The effect was donor-specific, since a combination of 25 μg α1–α3 H2Kd and 25 μg α1–α3 H2Dd proteins failed to affect survivals of third-party C3H (H2k k) heart allografts, namely 9.0 ± 0.0 days in treated versus 7.8 ± 0.5 days in untreated hosts. Thus, the combination of two H2K d and H2Dd proteins is more effective in prolonging allograft survival than a single protein produced in a bacterial expression system. A single PV injection (day 0) of 25 μg α1–α 2 H2Kd and 25 μg α1–α 2 H2Dd proteins to C57BL/10 mice prolonged the survival of BALB/c heart allografts to 22.4 ± 4.5 days. Within a WF to ACI rat heart allograft system, a single PV injection of 20 μg 70–77 u-RT1.Aa induced specific tolerance of allografts. This therapy could be combined with CsA to induce transplantation tolerance. However, combination of 70–77u-RT1.Aa with CTLA4Ig, rapamycin, or AG-490 effectively blocked the induction of transplantation tolerance. Tolerance generated by allochimeric protein could be adoptively transferred to naive recipients. Intragraft cytokine mRNA levels showed a bias towards a Th2-type phenotype. Additionally, studies of cytokine signaling and activation of transcription factors revealed a requirement that these pathways remain available for signaling in order for transplantation tolerance to occur. These studies suggest that the generation of regulatory cells are required for the induction of transplantation tolerance through the use of allochimeric proteins. ^

Supporting Whole-Class Collaborative Inquiry in a Secondary Mathematics Classroom

Recent mathematics education reform efforts call for the instantiation of mathematics classroom environments where students have opportunities to reason and construct their understandings as part of a community of learners. Despite some successes, traditional models of instruction still dominate the educational landscape. This limited success can be attributed, in part, to an underdeveloped understanding of the roles teachers must enact to successfully organize and participate in collaborative classroom practices. Towards this end, an in-depth longitudinal case study of a collaborative high school mathematics classroom was undertaken guided by the following two questions: What roles do these collaborative practices require of teacher and students? How does the community’s capacity to engage in collaborative practices develop over time? The analyses produced two conceptual models: one of the teacher’s role, along with specific instructional strategies the teacher used to organize a collaborative learning environment, and the second of the process by which the class’s capacity to participate in collaborative inquiry practices developed over time.

Effect of thiazolidinedione, a class of anti-diabetic drugs, on the mouse skin tumorigenesis

Thiazolidinediones (TZDs), a novel class of anti-diabetic drugs, have been known as ligands of peroxisome proliferator-activated receptor γ (PPARγ), a transcription factor that belongs to the nuclear receptor superfamily. These synthetic compounds improve insulin sensitivity in patients with type II diabetes likely through activating PAPRγ. Interestingly, they were also shown to inhibit cell growth and proliferation in a wide variety of tumor cell lines. The aim of this study is to assess the potential use of TZDs in the prevention of carcinogenesis using mouse skin as a model. ^ We found that troglitazone, one of TZD drugs, strongly inhibited cultured mouse skin keratinocyte proliferation as demonstrated by [3H]thymidine incorporation assay. It also induced a cell cycle G1 phase arrest and inhibited expression of cell cycle proteins, including cyclin D1, cdk2 and cdk4. Further experiments showed that PPARγ expression in keratinocytes was surprisingly undetectable in vitro or in vivo. Consistent with this, no endogenous PPARγ function in keratinocytes was found, suggesting that the inhibition of troglitazone on keratinocyte proliferation and cell cycle was PPARγ-independent. We further found that troglitazone inhibited insulin/insulin growth factor I (IGF-1) mitogenic signaling, which may explains, at least partly, its inhibitory effect on keratinocyte proliferation. We showed that troglitazone rapidly inhibited IGF-1 induced phosphorylation of p70S6K by mammalian target of rapamycin (mTOR). However, troglitazone did not directly inhibit mTOR kinase activity as shown by in vitro kinase assay. The inhibition of p70S6K is likely to be the result of strong activation of AMP activated protein kinase (AMPK) by TZDs. Stable expression of a dominant negative AMPK in keratinocytes blocked the inhibitory effect of troglitazone on IGF-1 induced phosphorylation of p70S6K. ^ Finally, we found that dietary TZDs inhibited by up to 73% mouse skin tumor development promoted by elevated IGF-1 signaling in BK5-IGF-1 transgenic mice, while they had no or little effect on skin tumor development promoted by 12-O-tetradecanoylphorbol-13-acetate (TPA) or ultraviolet (UV). Since IGF-1 signaling is frequently found to be elevated in patients with insulin resistance and in many human tumors, our data suggest that TZDs may provide tumor preventive benefit particularly to these patients. ^

Report on the practice based culminating experience as teaching assistant for "Pathology and Public Health" class taught at University of Texas School of Public Health, Houston

Objectives. The objectives of this report were to describe current best standards in online education, class competencies, class objectives, class activities and to compare the class competencies, objectives and activities undertaken with the current best practices in online teaching and to provide a list of recommendations based on the most efficacious practices. ^ Methods. Utilizing the key words- online teaching, national standards, quality, online courses, I: (1) conducted a search on Google to find the best standard for quality online courses; the search yielded National Standards for Quality Online Teaching as the gold standard in online course quality; (2) specified class objectives and competencies as well as major activities undertaken as a part of the class. Utilizing the Southern Regional Education Board evaluation checklist for online courses, I: (1) performed an analysis comparing the class activities, objectives, and competencies with the current best standards; (2) utilized the information obtained from the analysis and class experiences to develop recommendations for the most efficacious online teaching practices. ^ Results. The class met the criteria set by the Southern Regional Education Board for evaluating online classes completely in 75%, partially in 16% and did not meet the criteria in 9% cases. The majority of the parameters in which the class did not meet the standards (4 of 5) were due to technological reasons beyond the scope of the class instructor, teaching assistant and instructional design. ^ Discussion. Successful online teaching requires awareness of technology, good communication, methods, collaboration, reflection and flexibility. Creation of an online community, engaging online learners and utilizing different learning styles and assessment methods promote learning. My report proposes that online teaching should actively engage the students and teachers with multiple interactive strategies as evidenced from current best standards of online education and my “hands-on” work experience. ^ Conclusion. The report and the ideas presented are intended to create a foundation for efficacious practice on the online teaching platform. By following many of the efficacious online practices described in the report and adding from their own experiences, online instructors and teaching assistants can contribute to effective online learning. ^

THE CYTOPLASMIC TAIL OF MHC CLASS I MOLECULES PLAYS A CRITICAL ROLE IN DENDRITIC CELL-INDUCED T CELL IMMUNITY

The presentation of MHC class I (MHC-I)/peptide complexes by dendritic cells (DCs) is critical for the maintenance of central tolerance to self and for the regulation of cytotoxic T lymphocytes (CTL)-mediated adaptive immune responses against pathogens and cancer cells. Interestingly, several findings have suggested that the cytoplasmic tail of MHC class I plays a functional role in the regulation of CTL immune responses. For example, our previous studies demonstrated that exon 7-deleted MHC-I molecules not only showed extended DC cell surface half-lives but also induced significantly increased CTL responses to viral challange invivo. Although exon 7-deleted variant of MHC-I does not occur naturally in humans, the animal studies prompted us to examine whether exon 7-deleted MHC-I molecules could generate augmented CTL responses in a therapeutic DC-based vaccine setting. To examine the stimulatory capacity of exon 7-deleted MHC-I molecules, we generated a lentivirus-mediated gene transfer system to induce the expression of different MHC-I cytoplasmic tail isoforms in both mouse and human DCs. These DCs were then used as vaccines in a melanoma mouse tumor model and in a human invitro co-culture system. In this thesis, we show that DCs expressing exon 7-deleted MHC-I molecules, stimulated remarkably higher levels of T-cell cytokine production and significantly increased the proliferation of meanoma-specific (Pmel-1) T cells compared with DCs expressing wild type MHC-I. We also demonstrate that, in combination with adoptive transfer of Pmel-1 T-cell, DCs expressing exon 7-deleted Db molecules induced greater anti-tumor responses against established B16 melanoma tumors, significantly extending mouse survival as compared to DCs expressing wild-type Db molecules. Moreover, we also observed that human DCs expressing exon 7-deleted HLA-A2 molecules showed similarly augmented CTL stimulatory ability. Mechanistic studies suggest that exon 7-deleted MHC-I molecules showed impaired lateral membrane movement and extended cell surface half-lives within the DC/T-cell interface, leading to increased spatial availability of MHC-I/peptide complexes for recognition by CD8+ T cells. Collectively, these results suggesr that targeting exon 7 within the cytoplasmic tail of MHC-I molecules in DC vaccines has the potential to enhance CD8+ T cell stimulatory capacity and improve clinical outcomes in patients with cancer or viral infections.

Importance of antibiotic class to the development of Clostridium difficile infection (CDI) in adults: A systematic review

C. difficile causes gastrointestinal infections in humans, including severe diarrhea. It is implicated in 20%-30% of cases of antibiotic-associated diarrhea, in 50%-70% of cases of antibiotic-associated colitis, and in >90% of cases of antibiotic-associated pseudomembranous colitis. Exposure to antimicrobial agent, hospitalization and age are some of the risk factors that predispose to CDI. Virtually all hospitalized patients with nosocomially-acquired CDI have a history of treatment with antimicrobials or neoplastic agent within the previous 2 months. The development of CDI usually occurs during treatment with antibiotics or some weeks after completing the course of the antibiotics. ^ After exposure to the organism (often in a hospital), the median incubation period is less than 1 week, with a median time of onset of 2days. The difference in the time between the use of antibiotic and the development of the disease relate to the timing of exogenous acquisition of C. difficile. ^ This paper reviewed the literature for studies on different classes of antibiotics in association with the rates of primary CDI and RCDI from the year 1984 to 2012. The databases searched in this systematic review were: PubMed (National Library of Medicine) and Medline (R) (Ovid). RefWorks was used to store bibliographic data. ^ The search strategy yielded 733 studies, 692 articles from Ovid Medline (R) and 41 articles from PubMed after removing all duplicates. Only 11 studies were included as high quality studies. Out of the 11 studies reviewed, 6 studies described the development of CDI in non-CDI patients taking antibiotics for other purposes and 5 studies identified the risk factors associated with the development of recurrent CDI after exposure to antibiotics. ^ The risk of developing CDI in non-CDI patients receiving beta lactam antibiotics was 2.35%, while fluoroquinolones, clindamycin/macrolides and other antibiotics were associated with 2.64%, 2.54% and 2.35% respectively. Of those who received beta lactam antibiotic, 26.7% developed RCDI, while 36.8% of those who received any fluoroquinolone developed RCDI, 26.5% of those who received either clindamycin or macrolides developed RCDI and 29.1% of those who received other antibiotics developed RCDI. Continued use of non-C. difficile antibiotics especially fluoroquinolones was identified as an important risk factor for primary CDI and recurrent CDI. ^

A Novel Mechanism of Skin Tumor Promotion Involving Interferon-gamma (IFNγ)/Signal Transducer and Activator of Transcription-1 (Stat1) Signaling in Epidermis

The JAK-STAT pathway is a major signaling pathway involved in many biological processes including proliferation, apoptosis, and differentiation. Aberrant expression of STATs has been reported in multiple human cancers and murine mouse models of tumorigenesis. Previous studies from our lab and others have established a critical role for Stat3 in epithelial tumorigenesis, but the role of Stat1 is largely unknown. The current study was designed to explore the role of Stat1 during multistage skin carcinogenesis. Topical treatment with both TPA and the anthrone derivative chrysarobin (CHRY) led to rapid phosphorylation of Stat1 on both tyrosine (Tyr701) and serine (Ser727) residues in epidermis. CHRY treatment also led to upregulation of unphosphorylated Stat1 (uStat1) at later time points. In addition, CHRY treatment also led to upregulation of IRF-1 mRNA and protein which was dependent on Stat1. Further analyses demonstrated that topical treatment with CHRY but not TPA upregulated interferon-gamma (IFNg) mRNA in the epidermis and that the induction of both IRF-1 and uStat1 was dependent on IFNg signaling. Stat1 deficient (Stat1-/-) mice were highly resistant to skin tumor promotion by CHRY. In contrast, the tumor response (in terms of both papillomas and squamous cell carcinomas) was similar in Stat1-/- mice and wild-type littermates with TPA as the promoter. Histological evaluation of the proliferative response confirmed the data obtained from the tumor study for both TPA and CHRY. In addition, maximal induction of both cyclooxygenase-2 and inducible nitric oxide synthase in epidermis following treatment with CHRY was also dependent on the presence of functional Stat1. Following CHRY treatment, Stat1-/- mice exhibited reduced macrophage infiltration and reduced production of many immune cell derived chemokines/cytokines. These studies define a novel mechanism associated with skin tumor promotion by the anthrone class of tumor promoters involving upregulation of IFNg signaling in the epidermis and downstream signaling through activated (phosphorylated) Stat1 and subsequent upregulation of IRF-1 and uStat1.

Binding of the HIV-1 gp120 -V3 to cellular glycosphingolipids is necessary for CXCR4 recruitment and infection

Infection by human immunodeficiency virus type 1 (HIV-1) is a multi-step process, and detailed analyses of the various events critical for productive infection are necessary to clearly understanding the infection process and identifying novel targets for therapeutic interventions. Evidence from this study reveals binding of the viral envelope protein to host cell glycosphingolipids (GSLs) as a novel event necessary for the orderly progression of the host cell-entry and productive infection by HIV-1. Data obtained from co-immunoprecipitation analyses and confocal microscopy showed that the ability of viral envelope to interact with the co-receptor CXCR4 and productive infection of HIV-1 were inhibited in cells rendered GSL-deficient, while both these activities were restored after reconstitution of the cells with specific GSLs like GM3. Furthermore, evidence was obtained using peptide-inhibitors of HIV-1 infection to show that binding of a specific region within the V3-loop of the envelope protein gp120 to the host cell GSLs is the trigger necessary for the CD4-bound gp120 to recruit the CXCR4 co-receptor. Infection-inhibitory activity of the V3 peptides was compromised in GSL-deficient cells, but could be restored by reconstitution of GSLs. Based on these findings, a revised model for HIV-1 infection is proposed that accounts for the established interactions between the viral envelope and host cell receptors while enumerating the importance of the new findings that fill the gap in the current knowledge of the sequential events for the HIV-1 entry. According to this model, post-CD4 binding of the HIV-1 envelope surface protein gp120 to host cell GSLs, mediated by the gp120-V3 region, enables formation of the gp120-CD4-GSL-CXCR4 immune-complex and productive infection. The identification of cellular GSLs as an additional class of co-factors necessary for HIV-1 infection is important for enhancing the basic knowledge of the HIV-1 entry that can be exploited for developing novel antiviral therapeutic strategies. ^

(Table 1) Iceberg characteristics and start and end date and location of iceberg buoys in the Weddell Sea

The drift of 52 icebergs tagged with GPS buoys in the Weddell Sea since 1999 has been investigated with respect to prevalent drift tracks, sea ice/iceberg interaction, and freshwater fluxes. Buoys were deployed on small- to medium-sized icebergs (edge lengths ? 5 km) in the southwestern and eastern Weddell Sea. The basin-scale iceberg drift of this size class was established. In the western Weddell Sea, icebergs followed a northward course with little deviation and mean daily drift rates up to 9.5 ± 7.3 km/d. To the west of 40°W the drift of iceberg and sea ice was coherent. In the highly consolidated perennial sea ice cover of 95% the sea ice exerted a steering influence on the icebergs and was thus responsible for the coherence of the drift tracks. The northward drift of buoys to the east of 40°W was interrupted by large deviations due to the passage of low-pressure systems. Mean daily drift rates in this area were 11.5 ± 7.2 km/d. A lower threshold of 86% sea ice concentration for coherent sea ice/iceberg movement was determined by examining the sea ice concentration derived from Special Sensor Microwave Imager (SSM/I) and Advanced Microwave Scanning Radiometer for EOS (AMSR-E) satellite data. The length scale of coherent movement was estimated to be at least 200 km, about half the value found for the Arctic Ocean but twice as large as previously suggested. The freshwater fluxes estimated from three iceberg export scenarios deduced from the iceberg drift pattern were highly variable. Assuming a transit time in the Weddell Sea of 1 year, the iceberg meltwater input of 31 Gt which is about a third of the basal meltwater input from the Filchner Ronne Ice Shelf but spreads across the entire Weddell Sea. Iceberg meltwater export of 14.2 × 103 m3 s?1, if all icebergs are exported, is in the lower range of freshwater export by sea ice.

Table 1 Microorganisms, identified with DOGE analysis. 16S rDNA sequences, obtained from sequencing of DOGE bands, were assigned to the corresponding microorganisms

Microorganisms play an important role in the transformation of material within the earth's crust. The storage of CO2 could affect the composition of inorganic and organic components in the reservoir, consequently influencing microbial activities. To study the microbial induced processes together with geochemical, petrophysical and mineralogical changes, occurring during CO2 storage, long-term laboratory experiments under simulated reservoir P-T conditions were carried out. Clean inner core sections, obtained from the reservoir region at the CO2 storage site in Ketzin (Germany) from a depth of about 650 m, were incubated in high pressure vessels together with sterile synthetic formation brine under in situ P-T conditions of 5.5 MPa and 40°C. A 16S rDNA based fingerprinting method was used to identify the dominant species in DNA extracts of pristine sandstone samples. Members of the alpha- and beta-subdivisions of Proteobacteria and the Actinobacteria were identified. So far sequences belonging to facultative anaerobic, chemoheterotrophic bacteria (Burkholderia fungorum, Agrobacterium tumefaciens) gaining their energy from the oxidation of organic molecules and a genus also capable of chemolithoautotrophic growth (Hydrogenophaga) was identified. During CO2 incubation minor changes in the microbial community composition were observed. The majority of microbes were able to adapt to the changed conditions. During CO2 exposure increased concentrations of Ca**2+, K**+, Mg**2+ and SO4**2- were observed. Partially, concentration rises are (i) due to equilibration between rock pore water and synthetic brine, and (ii) between rock and brine, and are thus independent on CO2 exposure. However, observed concentrations of Ca**2+, K**+, Mg**2+ are even higher than in the original reservoir fluid and therefore indicate mineral dissolution due to CO2 exposure.

(Table 1) Age, fork length, and tissue d15N and element concentration of arctic char (Salvelinus alpinus) from Lake Hazen

Arctic char (Salvelinus alpinus L.), the top predator in High Arctic lakes, often is used as a bioindicator of Hg contamination in Arctic aquatic ecosystems. The present study investigated effects of trophic position, size, and age of Arctic char in Lake Hazen, the largest lake in the Canadian High Arctic (81°50'N, 70°25'W), on Hg bioaccumulation. In addition, several essential (Se, K) and nonessential elements (Tl, Cs) in char muscle tissue were examined to compare their behavior to that of Hg. Trophic position of Arctic char was identified by stable isotope (d15N) signature. Temporal trends of Hg from seven sampling campaigns over a 16-year period (1990-2006) were investigated for the overall data and for one trophic class. Concentrations of Hg were not correlated with age but were positively related to fork length and trophic position. Large char with greater d15N signatures (>12 per mil) had larger Hg concentrations (0.09-1.63 µg/g wet wt) than small char with smaller d15N signatures (<12 per mil, 0.03-0.32 µg/g wet wt), indicating that Hg concentrations increased with trophic position. Nonessential Cs and Tl showed relationships to age, length, and trophic position similar to those of Hg, indicating their potential to bioaccumulate and biomagnify. Essential Se and K did not show these relationships. Concentrations of Hg were adjusted using d15N, leading to less within-year variability and a more consistent temporal trend. The d15N-adjusted trend showed no decline of Hg in Arctic char from Lake Hazen (1990-2006) in the overall data set and in the small morphotype. Trends for the same period before the adjustment were not significant for the overall data set, but a slight decrease was apparent in the small morphotype. The results confirm the need to consider trophic position and fish size when monitoring temporal trends of Hg, particularly for species with different morphotypes.

(Table 1) Concentration of PCB and other contaminants in blood plasma of polar bears (Ursus maritimus) from Svalbard

Persistent chemicals accumulate in the arctic environment due to their chemical reactivity and physicochemical properties and polychlorinated biphenyls (PCBs) are the most concentrated pollutant class in polar bears (Ursus maritimus). Metabolism of PCB and polybrominated biphenyl ether (PBDE) flame-retardants alter their toxicological properties and these metabolites are known to interfere with the binding of thyroid hormone (TH) to transthyretin (TTR) in rodents and humans. In polar bear plasma samples no binding of [125I]-T4 to TTR was observed after incubation and PAGE separation. Incubation of the plasma samples with [14C]-4-OH-CB107, a compound with a higher binding affinity to TTR than the endogenous ligand T4 resulted in competitive binding as proven by the appearance of a radio labeled TTR peak in the gel. Plasma incubation with T4 up to 1 mM, a concentration that is not physiologically relevant anymore did not result in any visible competition. These results give evidence that the binding sites on TTR for T4 in wild living polar bears are completely saturated. Such saturation of binding sites can explain observed lowered levels of THs and could lead to contaminant transport into the developing fetus.