Designing and characterizing hyperpolarizable silicon nanoparticles for magnetic resonance imaging

Thesis (S.B.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2008.Includes bibliographical references (p. 37-41).Magnetic Resonance Imaging (MRI) is one of the most powerful noninvasive tools for diagnosing human disease, but its utility is limited because current contrast agents are ineffective when imaging air-tissue interfaces, in regions with low signal-tonoise ratios, or in areas that undergo motion, like the heart and bowel. A technique called dynamic nuclear polarization can be used to hyperpolarize nuclei and achieve dramatic MRI signal enhancement with minimal background noise. It has been shown that ballmilled silicon nanoparticles have the advantageous properties of hyperpolarizability and biodegradability, but in vivo utilization requires the modification of the particle surface to prevent aggregation that leads to very fast removal from circulation through phagocytosis by the liver, spleen, and lymph nodes. This thesis describes a method to functionalize hyperpolarizable silicon nanoparticles using silane chemistry and coating by poly(ethylene glycol). The particles were characterized using dynamic light scattering, scanning electron microscopy, and laser Doppler electrophoresis. The extent of amination was quantified using a fluorescamine assay, and stability was assessed by visualizing flocculation and measuring aggregation in different solvents. The functionalized particles were stable in solutions that resemble physiological conditions. These silicon nanoparticles can potentially be used for in vivo cancer imaging to enable early diagnoses and assist with clinical decision-making through disease monitoring.by Melis Nuray Anahtar.S.B

The Contribution of Cervicovaginal Microbiota and Hormonal Contraceptives to Genital Inflammation and HIV Acquisition Risk

The HIV epidemic persists in many parts of the world, with the majority of new infections occurring through the female genital tract (FGT). The permissiveness of the genital mucosa to HIV is modulated by the integrity of the epithelial barrier, the presence of pro-inflammatory cytokines, and the frequency of CCR5+CD4+ T cell targets. Here we focus on two biological perturbations: endogenous alterations in cervicovaginal bacteria and exogenous injectable progestin-only contraceptives (IPCs). We examine their effects on the genital mucosal environment and their link to HIV susceptibility in a cohort of young South African women. We first sought to determine how genital microbiota modulate host inflammatory responses. The existing paradigm is that vaginal monocolonization by Lactobacillus is normal, and encroachment by other bacteria is pathologic. By characterizing cervicovaginal bacterial communities in 94 South African women using 16S rRNA and shotgun sequencing, we found that the majority of participants had low Lactobacillus abundance and high ecological diversity. One diverse Prevotella-containing community type strongly correlated with increased concentrations of multiple genital pro-inflammatory cytokines in vivo. We found that these cytokines were produced by epithelial cells and antigen presenting cells via different bacterial sensing mechanisms. Our results identify specific bacterial species that alter the inflammatory state of the FGT and may more broadly impact reproductive health in women. We also investigated the immunological effects of IPCs, the most common form of birth control in sub-Saharan Africa. Although highly effective as a contraceptive, IPCs are controversially associated with increased HIV susceptibility by an unclear mechanism. We found that IPC users had a 5.5-fold higher risk of acquiring HIV than women not using family planning (p=0.0031, 95% CI: 1.733 – 16.80). Phenotypic cellular analysis revealed that IPC users also had a significantly higher frequency of activated HIV target cells in the cervix. Since the availability of target cells in the genital mucosa enables early viral replication, recruitment or retention of these cells by IPCs may explain the observed increased HIV acquisition rates. Furthermore, IPC use was not associated with differences in genital cytokine levels, indicating that cervicovaginal bacteria and exogenous progesterone increase HIV susceptibility by unique pathways.Medical Science

Autologous Platelet Collection and Storage to Support Thrombocytopenia in a Leukemia Patient with Platelet Alloimmunization Undergoing Chemotherapy

ABSTRACT Platelet alloimmunization occurs frequently in multitransfused patients. To prevent the posttransfusion complications donor white blood cells (WBCs) must be removed from the platelet concentrates (PC). But sometimes the centrifugation and filtration of WBC is not enough and in such conditions HLA-matched PC are recommended. Cryopreservation of autologous PC offers a potential solution to this problem. We investigated the feasibility of supporting the aplastic period after chemotherapy in a 54-year-old leukemia patient with autologous platelets collected by apheresis and cryopreserved. Our case report demonstrates that autologous apheresis platelet transfusion might be a feasible approach to high risk patients with bleeding tendencies. Key Words: Blood transfusion autologous, Platelet, ‹mmunization, Transfusion, Allogeneic. ÖZET Kemoterapi Sonras› Trombosit Alloimmünizasyonu Geliflen Lösemili Bir Hastada Otolog Trombosit Aferezi ve Saklanmas› Trombosit alloimmünizasyonu çoklu transfüzyon yap›lan bireylerde karfl›lafl›lan önemli bir problemdir. Transfüzyon sonras› geliflecek komplikasyonlar› önlemek için transfüze edilecek üründeki lökositlerin uzaklaflt›r›lmas› gerekmektedir. Ancak bazen bu ifllem için uygulanan santrifügasyon ve filtrasyon yeterli olmamaktad›r, bu durumda HLA uyumlu trombosit transfüzyonu önerilmektedir. Bir baflka çözüm de kriyopreserve edilen otolog trombositlerin transfüzyonudur. Bu olgu sunumunda, indüksiyon tedavisi s›ras›nda trombosit refrakterli¤i geliflen akut lösemili bir olguda tromboferez sonras› kriyopreserve edilen otolog trombosit transfüzyonunun yarar› incelendi

Rapid, efficient functional characterization and recovery of HIV-specific human CD8+ T cells using microengraving

The nature of certain clinical samples (tissue biopsies, fluids) or the subjects themselves (pediatric subjects, neonates) often constrain the number of cells available to evaluate the breadth of functional T-cell responses to infections or therapeutic interventions. The methods most commonly used to assess this functional diversity ex vivo and to recover specific cells to expand in vitro usually require more than 106 cells. Here we present a process to identify antigen-specific responses efficiently ex vivo from 104–105 single cells from blood or mucosal tissues using dense arrays of subnanoliter wells. The approach combines on-chip imaging cytometry with a technique for capturing secreted proteins—called “microengraving”—to enumerate antigenspecific responses by single T cells in a manner comparable to conventional assays such as ELISpot and intracellular cytokine staining. Unlike those assays, however, the individual cells identified can be recovered readily by micromanipulation for further characterization in vitro. Applying this method to assess HIV-specific T cell responses demonstrates that it is possible to establish clonal CD8+ T-cell lines that represent the most abundant specificities present in circulation using 100- to 1,000-fold fewer cells than traditional approaches require and without extensive genotypic analysis a priori. This rapid (<24 h), efficient, and inexpensive process should improve the comparative study of human T-cell immunology across ages and anatomic compartments

Role of lactobacilli and lactoferrin in the mucosal cervicovaginal defense

Human lactoferrin is an iron-binding glycoprotein present at high concentrations in breast milk and colostrum. It is produced by many exocrine glands and widely distributed in a variety of body fluids. This protein has antimicrobial, immunomodulatory, antioxidant, and anticancer properties. Two important hLf receptors have been identified: LDL receptor related protein (LRP1), a low specificity receptor, and intelectin-1 (ITLN1), a high specificity receptor. No data are present on the role of hLf on the biliary epithelium. Our aims have been to evaluate the expression of Lf and its receptors in human and murine cholangiocytes and its effect on proliferation. Immunohistochemistry and immunofluorescence (IF) were conducted on human healthy and primary biliary cholangitis (PBC) liver samples as well as on liver samples obtained from normal and bile duct ligated (BDL) mice to evaluate the expression of Lf, LRP1 and ITLN1. Cell proliferation in vitro studies were performed on human cholangiocyte cell lines via 3-(4,5-dimetiltiazol-2-il)-2,5-diphenyltetrazolium assay as well as IF to evaluate proliferating cell nuclear antigen (PCNA) expression. Our results show that mouse and human cholangiocytes express Lf, LRP1 and ITLN1, at higher extent in cholangiocytes from BDL and PBC samples. Furthermore, the in vitro addition of bovine Lf (bLf) has a proliferative effect on human cholangiocyte cell line. The results support a proliferative role of hLf on the biliary epithelium; this pro-proliferative effect of hLf and bLf on cholangiocytes could be particularly relevant in human cholangiopathies such as PBC, characterized by cholangiocyte death and ductopenia

The interaction between vaginal microbiota, cervical length, and vaginal progesterone treatment for preterm birth risk

© The Author(s) 2017. Background: Preterm birth is the primary cause of infant death worldwide. A short cervix in the second trimester of pregnancy is a risk factor for preterm birth. In specific patient cohorts, vaginal progesterone reduces this risk. Using 16S rRNA gene sequencing, we undertook a prospective study in women at risk of preterm birth (n = 161) to assess (1) the relationship between vaginal microbiota and cervical length in the second trimester and preterm birth risk and (2) the impact of vaginal progesterone on vaginal bacterial communities in women with a short cervix. Results: Lactobacillus iners dominance at 16 weeks of gestation was significantly associated with both a short cervix < 25 mm (n = 15, P < 0.05) and preterm birth < 34+0 weeks (n = 18; P < 0.01; 69% PPV). In contrast, Lactobacillus crispatus dominance was highly predictive of term birth (n = 127, 98% PPV). Cervical shortening and preterm birth were not associated with vaginal dysbiosis. A longitudinal characterization of vaginal microbiota (< 18, 22, 28, and 34 weeks) was then undertaken in women receiving vaginal progesterone (400 mg/OD, n = 25) versus controls (n = 42). Progesterone did not alter vaginal bacterial community structure nor reduce L. iners-associated preterm birth (< 34 weeks). Conclusions: L. iners dominance of the vaginal microbiota at 16 weeks of gestation is a risk factor for preterm birth, whereas L. crispatus dominance is protective against preterm birth. Vaginal progesterone does not appear to impact the pregnancy vaginal microbiota. Patients and clinicians who may be concerned about "infection risk" associated with the use of a vaginal pessary during high-risk pregnancy can be reassured

Amygdala inputs to prefrontal cortex guide behavior amid conflicting cues of reward and punishment

Orchestrating appropriate behavioral responses in the face of competing signals that predict either rewards or threats in the environment is crucial for survival. The basolateral nucleus of the amygdala (BLA) and prelimbic (PL) medial prefrontal cortex have been implicated in reward-seeking and fear-related responses, but how information flows between these reciprocally connected structures to coordinate behavior is unknown. We recorded neuronal activity from the BLA and PL while rats performed a task wherein competing shock- and sucrose-predictive cues were simultaneously presented. The correlated firing primarily displayed a BLA→PL directionality during the shock-associated cue. Furthermore, BLA neurons optogenetically identified as projecting to PL more accurately predicted behavioral responses during competition than unidentified BLA neurons. Finally photostimulation of the BLA→PL projection increased freezing, whereas both chemogenetic and optogenetic inhibition reduced freezing. Therefore, the BLA→PL circuit is critical in governing the selection of behavioral responses in the face of competing signals.National Institutes of Health (U.S.) (Award 1R25-MH092912-01)National Institute of Mental Health (U.S.) (Grant R01- MH102441-01)National Institutes of Health (U.S.) (Award DP2- DK-102256-01

HIV-1 subtype C transmitted founders modulate dendritic cell inflammatory responses

Background Heterosexual transmission remains the main route of HIV-1 transmission and female genital tract (FGT) inflammation increases the risk of infection. However, the mechanism(s) by which inflammation facilitates infection is not fully understood. In rhesus macaques challenged with simian immunodeficiency virus, dendritic cell (DC) mediated recruitment of CD4+ T cells to the FGT was critical for infection. The aim of this study was to delineate the mechanisms underlying DC-mediated HIV infection by comparing chemokine and pro-inflammatory cytokine production in response to transmitted founder (TF) and chronic infection (CI) Envelope (Env) pseudotyped viruses (PSV). Results Monocyte-derived DCs (MDDCs) were stimulated with PSV and recombinant gp140 representing matched TF and CI pairs of four individuals and cytokine secretion measured by multiplex immuno-assay. We found that 4/9 Env induced robust MDDC inflammatory responses and of those, three were cloned from TFs. Overall, TF Env induced MDDCs from healthy donors to secrete higher concentrations of inflammatory cytokines and chemokines than those from CI, suggesting TF Env were better inducers of inflammation. Assessing the signalling pathway associated with inflammatory cytokines, we found that PSV of matched TF and CI variants and a gp140 clone activated ERK and JNK to similar levels. Recombinant soluble DC-SIGN inhibited cytokine release and activation of ERK by PSV, suggesting that Env-DC-SIGN binding was partly involved in MDDC stimulation. Therefore, Env clones might differentially stimulate MDDC immune responses via alternative, yet unidentified signalling pathways. Conclusion Overall, this could suggest that the genetics of the virus itself influences inflammatory responses during HIV infection. In the absence of pre-existing infections, induction of greater inflammatory response by TFs might favour virus survival within the healthy FGT by driving an influx of target cells to sites of infection while suppressing immune responses via IL-10

Genital inflammation undermines the effectiveness of tenofovir gel in preventing HIV acquisition in women.

CAPRISA, 2018.Abstract available in pdf