Versatility of M13 bacteriophage in medicine : vaccine storage and cancer diagnostics

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Materials Science and Engineering, 2007.Includes bibliographical references (p. 88-94).Two novel ways of engineering the filamentous bacteriophage, M13, for the prevention, diagnosis, and treatment of human disease are proposed. Both ways are founded on the unique structural properties of the M13 bacteriophage and the ability of its major and minor coat proteins, p3 and p8, to be manipulated to serve as virus-based multifunctional platforms. The first project addresses the problem of vaccine storage and the cold chain (requirement to store vaccines at 2-80C or lower). The need for refrigeration leads to high cost, difficult field delivery, and high potential for vaccine instability. By capitalizing on the liquid crystalline nature and unique diffraction patterns of phage films, we aim to encapsulate vaccines in a 3-D liquid crystalline matrix that would not only allow for stability at elevated temperatures but would also allow for easy detection of viability by using a laser light and noting the diffraction pattern. We chose luciferase as a model for a protein-based vaccine, and found several phage-borne peptide sequences with increased affinity to luciferase compared to controls. Two of these sequences, CKLHGTSRC and CTHKNQAC were chosen to form luciferase-encapsulated phage films. The second project addresses the need of more sensitive imaging techniques for early detection of cancer.(cont.) M13 bacteriophage were used in combination with quantum dots and magnetic nanoparticles as bigger and brighter markers for cancer lesions. A 100% expressed p8 library was created for screening against potential cancer markers and work will soon proceed with screening against several cancer cell lines. In addition, a Type 83 phage was created that had a sequence directed against vascular cell adhesion molecule-1 (VCAM-1) expressed on the p3 and a tri-glutamate sequence (E3) on the p8 that could bind well to positively charge molecules like amines. Successful attachment of amine-terminated CdSe/ZnS quantum dots to p8E3 phage (E3 on p8 and wildtype on p3) was shown and III-V quantum dots (GaN and InN) were water solubilized for use in vivo. The goal is to combine all parts and start in vivo testing and screening, as well as to expand our cancer targeting repertoire.by Amy Shi.S.M

Novel strategies for characterizing T Cell responses in SIV-infected rhesus monkeys

Thesis (S.M.)--Harvard-MIT Division of Health Sciences and Technology, 2009.Cataloged from PDF version of thesis.Includes bibliographical references (p. 103-114).Human Immunodeficiency Virus (HIV) is the cause of Acquired Immune Deficiency Syndrome (AIDS) and has killed over 25 million people since the disease was first recognized in 1981. As of 2007, 33 million people globally are infected with HIV and this number is growing. HIV infects and depletes CD4+ helper T cells, affecting the ability of the immune system to defend the host against common infections. While anti-retroviral therapy has decreased morbidity and mortality, these drugs are not curative. In addition, they are beyond the financial reach of many HIV infected patients. Thus, the development of strategies to control HIV spread is a high priority. The most relevant animal model for studying HIV is the Simian Immunodeficiency Virus (SIV) - infected rhesus monkey. While HIV research has focused on studying peripheral blood specimens, mucosal sites have recently been identified as a focal point for HIV replication and tissue destruction. They are usually the sites of primary infection in the setting of sexual transmission and they are also important sites of immune depletion. If methods for controlling the replication of the virus early after infection in mucosal sites are available, it may be possible to eliminate the virus prior to systemic spread. While strategies for generating strong neutralizing antibody responses have not yet been developed, emerging data suggest that CD8+ cytotoxic T cells can contribute substantially to early virus control. It is important to study CD8+ T cells in the setting of SIV infection in rhesus monkeys, particularly in mucosal sites, using functional as well as transcriptional assays.(cont.) One of the challenges in studying mucosal cellular immunity is the limited number of cells available in biopsies, making traditional assay systems such as flow cytometry very difficult to employ. Here, technologies for isolating rare cell populations and extracting RNA from these cells for gene expression analysis were developed. These technologies were then applied to peripheral blood specimens, looking at gene expression differences between virus-specific CD8+ T cells in Mamu-A*01+ and Mamu-A*02+ monkeys. The ultimate goal of these studies is to gain a better understanding of SIV immunopathogenesis (as a model for HIV immunopathogenesis) and to find a way to control or eliminate the virus.by Amy Shi.S.M

Astrobiological and Geological Implications of Convective Transport in Icy Outer Planet Satellites

The oceans of large icy outer planet satellites are prime targets in the search for extraterrestrial life in our solar system. The goal of our project has been to develop models of ice convection in order to understand convection as an astrobiologically relevant transport mechanism within icy satellites, especially Europa. These models provide valuable constraints on modes of surface deformation and thus the implications of satellite surface geology for astrobiology, and for planetary protection. Over the term of this project, significant progress has been made in three areas: (1) the initiation of convection in large icy satellites, which we find probably requires tidal heating; (2) the relationship of surface features on Europa to internal ice convection, including the likely role of low-melting-temperature impurities; and (3) the effectiveness of convection as an agent of icy satellite surface-ocean material exchange, which seems most plausible if tidal heating, compositional buoyancy, and solid-state convection work in combination. Descriptions of associated publications include: 3 published papers (including contributions to 1 review chapter), 1 manuscript in revision, 1 manuscript in preparation (currently being completed under separate funding), and 1 published popular article. A myriad of conference abstracts have also been published, and only those from the past year are listed

The natural history of EGFR and EGFRvIII in glioblastoma patients

BACKGROUND: The epidermal growth factor receptor (EGFR) is over expressed in approximately 50–60% of glioblastoma (GBM) tumors, and the most common EGFR mutant, EGFRvIII, is expressed in 24–67% of cases. This study was designed to address whether over expressed EGFR or EGFRvIII is an actual independent prognostic indicator of overall survival in a uniform body of patients in whom gross total surgical resection (GTR; ≥ 95% resection) was not attempted or achieved. METHODS: Biopsed or partially/subtotally resected GBM patients (N = 54) underwent adjuvant conformal radiation and chemotherapy. Their EGFR and EGFRvIII status was determined by immunohistochemistry and Kaplan-Meier estimates of overall survival were obtained. RESULTS: In our study of GBM patients with less than GTR, 42.6% (n = 23) failed to express EGFR, 25.9% (n = 14) had over expression of the wild-type EGFR only and 31.5 % (n = 17) expressed the EGFRvIII. Patients within groups expressing the EGFR, EGFRvIII, or lacking EGFR expression did not differ in age, Karnofsky Performance Scale (KPS) score, extent of tumor resection. They all had received postoperative radiation and chemotherapy. The median overall survival times for patients with tumors having no EGFR expression, over expressed EGFR only, or EGFRvIII were 12.3 (95% CI, 8.04–16.56), 11.03 (95% CI, 10.18–11.89) and 14.07 (95% CI, 7.39–20.74) months, respectively, log rank test p > 0.05). Patients with tumors that over expressed the EGFR and EGFRvIII were more likely to present with ependymal spread, 21.4% and 35.3% respectively, compared to those patients whose GBM failed to express either marker, 13.0%, although the difference was not statistically significant. There was no significant difference in multifocal disease or gliomatosis cerebri among EGFR expression groups. CONCLUSION: The over expressed wild-type EGFR and EGFRvIII are not independent predictors of median overall survival in the cohort of patients who did not undergo extensive tumor resection

Evidence for Class-Specific Factors in Immunoglobulin Isotype Switching

Immunoglobulin class switch recombination (SR) occurs by a B cell–specific, intrachromosomal deletional process between switch regions. We have developed a plasmid-based transient transfection assay for SR to test for the presence of transacting switch activities. The plasmids are novel in that they lack a eukaryotic origin of DNA replication. The recombination activity of these switch substrates is restricted to a subset of B cell lines that support isotype switching on their endogenous loci and to mitogen-activated normal splenic B cells. The factors required for extrachromosomal plasmid recombination are constitutively expressed in proliferating splenic B cells and in B cell lines capable of inducibly undergoing immunoglobulin SR on their chromosomal genes. These studies suggest that mitogens that induce switching on the chromosome induce accessibility rather than switch recombinase activity. Finally, we provide evidence for two distinct switching activities which independently mediate μ→α and μ→γ3 SR

Peptide nanofiber hydrogel adjuvanted live virus vaccine enhances cross-protective immunity to porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV) is prevalent in swine farms worldwide and is a major source of economic loss and animal suffering. Rapid genetic variation of PRRSV makes it difficult for current vaccines to confer protection against newly emerging strains. We recently demonstrated that a novel peptide nanofiber hydrogel (H9e) could act as a potent adjuvant for killed H1N1 vaccines. Therefore, the objective of this study was to evaluate H9e as an adjuvant for PRRSV modified live virus (MLV) vaccines. Pigs were vaccinated with Ingelvac PRRSV MLV with or without H9e adjuvant before being challenged with the VR-2332 (parental vaccine strain) or MN184A (genetically diverse strain) PRRSV. Pigs vaccinated with MLV+H9e had higher levels of circulating vaccine virus. More importantly, pigs vaccinated with MLV+H9e had improved protection against challenge by both PRRSV strains, as demonstrated by reduced challenge-induced viremia compared with pigs vaccinated with MLV alone. Pigs vaccinated with MLV+H9e had lower frequency of T-regulatory cells and IL-10 production but higher frequency of Th/memory cells and IFN-γ secretion than that in pigs vaccinated with MLV alone. Taken together, our studies suggest that the peptide nanofiber hydrogel H9e, when combined with the PRRSV MLV vaccine, can enhance vaccine efficacy against two different PRRSV strains by modulating both host humoral and cellular immune responses

Impact of crisaborole in patients with mild-to-moderate atopic dermatitis who received prior treatment

Topical treatments can provide relief with minimal adverse events (AEs) in patients with atopic dermatitis (AD). Crisaborole ointment 2% is a nonsteroidal phosphodiesterase inhibitor for the treatment of mild-to-moderate AD. The objective was to assess the efficacy and safety of crisaborole ointment in patients with AD who had received prior treatments of corticosteroids [topical corticosteroids (TCS) or systemic corticosteroids)], topical calcineurin inhibitors (TCI) or no prior treatments (treatment-naive). This was a post-hoc analysis of two identically designed, vehicle-controlled, randomized, double-blind, phase III studies of patients aged ≥2 years (ClinicalTrials.gov NCT02118766 and NCT02118792). Patients were assigned crisaborole or vehicle (2: 1) twice daily for 28 days and had a baseline Investigator’s Static Global Assessment (ISGA) score of mild (2) or moderate (3). Patients were divided into three subgroups: treatment-experienced patients who had received prior treatments of corticosteroids (systemic or dermatologic) or TCI; treatment-experienced patients who had received prior treatment with TCS or TCI; and treatment-naive patients who received no prior treatments within 90 days to screening. The primary endpoint was success in ISGA, defined as an ISGA score at day 29 of clear (0) or almost clear (1) with a ≥ 2-grade improvement from baseline. Additional endpoints included a Severity of Pruritus Scale (SPS) at week 4 (weekly average) of none (0) or mild (1) with a ≥ 1-grade improvement from baseline; changes in the Atopic Dermatitis Severity Index (ADSI), Dermatology Life Quality Index (DLQI), Children’s Dermatology Life Quality Index (CDLQI) and Dermatitis Family Impact Questionnaire (DFI) results were also assessed at day 29. AEs, including treatment-emergent AEs and serious AEs, were analysed. A significantly higher proportion of crisaborole-treated patients than vehicle-treated patients achieved success in ISGA in all subgroups [corticosteroids or TCI: 27.9% vs. 15.9% (P = 0.001); TCS or TCI: 27.4% vs. 14.7% (P = 0.001); treatment-naive: 35.0% vs. 26.8% (P = 0.017)]. SPS score 0/1 with a ≥ 1-grade improvement was also achieved by a significantly higher proportion of crisaborole-treated patients than vehicle-treated patients in all subgroups [corticosteroids or TCI: 35.1% vs. 14.9% (P \u3c 0.001); TCS or TCI: 34.5% vs. 13.5% (P \u3c 0.001); treatment-naive: 36.3% vs. 26.0% (P = 0.01)]. Changes in the least squares mean for ADSI, DLQI, CDLQI and DFI results were also significant for crisaborole- vs. vehicle-treated patients in all subgroups except for DLQI, DFI and ADSI (not examined) results for the treatment naive subgroup. Treatment-related AEs were infrequent and mild to moderate in severity. Crisaborole demonstrated a favourable efficacy and safety profile in both treatment-naive and treatment-experienced patients with AD

TLR2 and Caspase-1 Signaling are Critical for Bacterial Containment But Not Clearance During Craniotomy-Associated Biofilm Infection

BACKGROUND: A craniotomy is required to access the brain for tumor resection or epilepsy treatment, and despite precautionary measures, infectious complications occur at a frequency of 1-3%. Approximately half of craniotomy infections are caused by Staphylococcus aureus (S. aureus) that forms a biofilm on the bone flap, which is recalcitrant to antibiotics. Our prior work in a mouse model of S. aureus craniotomy infection revealed a critical role for myeloid differentiation factor 88 (MyD88) in bacterial containment and pro-inflammatory mediator production. Since numerous receptors utilize MyD88 as a signaling adaptor, the current study examined the importance of Toll-like receptor 2 (TLR2) and TLR9 based on their ability sense S. aureus ligands, namely lipoproteins and CpG DNA motifs, respectively. We also examined the role of caspase-1 based on its known association with TLR signaling to promote IL-1β release. METHODS: A mouse model of craniotomy-associated biofilm infection was used to investigate the role of TLR2, TLR9, and caspase-1 in disease progression. Wild type (WT), TLR2 knockout (KO), TLR9 KO, and caspase-1 KO mice were examined at various intervals post-infection to quantify bacterial burden, leukocyte recruitment, and inflammatory mediator production in the galea, brain, and bone flap. In addition, the role of TLR2-dependent signaling during microglial/macrophage crosstalk with myeloid-derived suppressor cells (MDSCs) was examined. RESULTS: TLR2, but not TLR9, was important for preventing S. aureus outgrowth during craniotomy infection, as revealed by the elevated bacterial burden in the brain, galea, and bone flap of TLR2 KO mice concomitant with global reductions in pro-inflammatory mediator production compared to WT animals. Co-culture of MDSCs with microglia or macrophages, to model interactions in the brain vs. galea, respectively, also revealed a critical role for TLR2 in triggering pro-inflammatory mediator production. Similar to TLR2, caspase-1 KO animals also displayed increased S. aureus titers coincident with reduced pro-inflammatory mediator release, suggestive of pathway cooperativity. Treatment of caspase-1 KO mice with IL-1β microparticles significantly reduced S. aureus burden in the brain and galea compared to empty microparticles, confirming the critical role of IL-1β in limiting S. aureus outgrowth during craniotomy infection. CONCLUSIONS: These results demonstrate the existence of an initial anti-bacterial response that depends on both TLR2 and caspase-1 in controlling S. aureus growth; however, neither pathway is effective at clearing infection in the WT setting, since craniotomy infection persists when both molecules are present

Detection of skewed X-chromosome inactivation in Fragile X syndrome and X chromosome aneuploidy using quantitative melt analysis.

Methylation of the fragile X mental retardation 1 (FMR1) exon 1/intron 1 boundary positioned fragile X related epigenetic element 2 (FREE2), reveals skewed X-chromosome inactivation (XCI) in fragile X syndrome full mutation (FM: CGG > 200) females. XCI skewing has been also linked to abnormal X-linked gene expression with the broader clinical impact for sex chromosome aneuploidies (SCAs). In this study, 10 FREE2 CpG sites were targeted using methylation specific quantitative melt analysis (MS-QMA), including 3 sites that could not be analysed with previously used EpiTYPER system. The method was applied for detection of skewed XCI in FM females and in different types of SCA. We tested venous blood and saliva DNA collected from 107 controls (CGG < 40), and 148 FM and 90 SCA individuals. MS-QMA identified: (i) most SCAs if combined with a Y chromosome test; (ii) locus-specific XCI skewing towards the hypomethylated state in FM females; and (iii) skewed XCI towards the hypermethylated state in SCA with 3 or more X chromosomes, and in 5% of the 47,XXY individuals. MS-QMA output also showed significant correlation with the EpiTYPER reference method in FM males and females (P < 0.0001) and SCAs (P < 0.05). In conclusion, we demonstrate use of MS-QMA to quantify skewed XCI in two applications with diagnostic utility