Browsing by Author "Luchini, Alessandra"
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Item Affinity Functionalized Strings for Blood PurificationCachaco, Silvia; Luchini, AlessandraPathogen reduction is a proactive approach to maximize safety of blood transfusion. Methods of bacterial reduction are available for plasma and platelets but not for red blood cell concentrates and whole blood. We addressed this technology gap by using a novel approach consisting of molecular probes immobilized on nylon filaments (affinity networks) as a means to capture bacteria in suspension. We dyed nylon filaments with 24 dyes with different chemical properties, including acidic, basic, fast, metallic, hydrophobic, and uncharged/polar dyes. We established quality control measures of acceptable ranges of dye incorporation in the nylon network to ensure reproducibility. We performed incubations and microbiology cultures to assess the depletion capability of different affinity networks. Four affinity networks incorporating basic and hydrophobic dyes (pinacyanol chloride, reactive blue 21, sudan IV, alizarin cyanin) depleted more than 90% of E. coli suspended in phosphate buffer saline (PBS, 10 and 100 CFU/mL). Safranin O, methylene blue, alcian blue pyridine variant, and reactive blue 21 dyed nylon networks removed more than 90% of S. epidermidis from PBS (10 and 100 CFU/mL). In several instances, affinity networks removed all bacteria present in suspension. Affinity networks efficiently captured >90% S. epidermidis spiked in plasma at a 10 and 100 CFU/mL concentration, and the combination of sudan IV, alcian blue pyridine variant, and reactive blue 21 functionalized affinity networks had synergistic effects. Affinity network sequestration of lipoteichoic acid (LTA), a major component of gram-positive bacterium cell wall, spiked in model solutions, suggested that LTA is involved in the binding and capture of bacteria. In conclusion, affinity networks functionalized with synthetic dyes is a promising medium to capture bacteria from suspensions and from biological matrices for future application as a pathogen reduction technology. Future work will include a prototype production for a pathogen reduction device.Item Developing Low-Density Lipoprotein Receptor-Related Protein-1 Agonists as a Therapeutic Strategy in Allergic Inflammatory Diseases(2022) Austin, Dana; Luchini, Alessandra; Gelber, CohavaAllergic inflammatory diseases such as atopic dermatitis, eosinophilic esophagitis (EoE) and asthma are a consequence of persistent exposure to allergens leading to impaired innate immune mechanisms and dysregulated immune responses. A large unmet need exists for an effective treatment that is devoid of potentially serious side effects and without immunosuppressive effects. We have previously developed a family of first-in-class drugs derived from naturally occurring Serine Protease Inhibitors (SERPINS) that target Low-Density Lipoprotein Receptor-Related Protein-1 (LRP-1), a homeostatic receptor that regulates a multitude of critical functions of the immune response. The lead peptide, SP16, is a short (17mer) modified peptide derivative of Alpha-1 Antitrypsin (AAT). Recent work has shown that both AAT and LRP-1 play a role in alleviating allergen-driven inflammation. In this study, we have designed a series of SP16-derivative analogs modeled on the putative LRP-1 binding site, with the goal of developing these peptide therapeutics for inflammatory diseases. We have methodically screened these analogs for their ability to inhibit inflammation, refining the analog design based on structure-activity relationships. In three tissue specific cell lines, analogs showed increased potency, with dramatically improved effective concentrations (EC50s) compared to SP16, and no toxicity, thus having the potential for an increased therapeutic window. In-vitro, ELISA based LRP-1 binding assays showed target engagement. Lead analogs, screened in a rapid in-vivo mouse model of acute inflammation, exhibited significant anti-inflammatory function comparable to SP16. The analogs demonstrated improved pharmacokinetic properties and potential for a wider range of administration routes, including oral. Finally, a few select analogs, alongside SP16, were tested for their ability to inhibit TH2 mediated responses in mouse models of atopic dermatitis and asthma. Both SP16 and analogs showed significant amelioration of disease phenotypes, including thymic stromal lymphopoietin (TSLP) inhibition. Overall, the developmental work has defined two new lead SERPIN-derived LRP-1 agonists for inflammatory diseases and provided a new avenue of SP16 development in allergic inflammation.Item Effects of Hydrogel Nanoparticles as a Novel Adjuvant for a Purified Whole Inactivated Chikungunya Virus VaccineJackson, Kassandra; Jackson, Kassandra; Luchini, AlessandraThis thesis describes a novel nanotrap nanoparticle adjuvant for a purified whole inactivated Chikungunya CHIK virus vaccine and its immune response and level of toxicity. For a vaccine to be good candidate it must be able to invoke a strong immune response to the viral antigen without causing damage to the recipient. Most vaccines are formulated with an adjuvant, which is anything that has the ability to bolster the immune response to the vaccine. Nanoparticle pose as a promising alternative to the commonly used alum adjuvant. The novel chemistry of the nanoparticles used in this study allows them to deliver the drug to the desired area, gradually release their cargo, and stabilize and protect the vaccine. Following a 28-day mouse trail, blood sera from the placebo group, non-adjuvanted CHIK vaccine group and NP-adjuvanted CHIK vaccine group were tested for neutralizing antibodies. Each vaccine group was also tested for signs of toxicity. The neutralizing titers from the NP-adjuvanted vaccine group was substantially equivalent to the non-adjuanted vaccine group. Moreover, the author showed that the NPs were able to capture whole CHIK virus, remain adsorbed to the virus for an extended period of time and that the NPs were non-toxic.Item Investigation of Small Peptide Inhibitors on PD-1 PD-L1 Protein-Protein InteractionsDey, Douglass; Dey, Douglass; Luchini, AlessandraCancer is a complex disease in which abnormal cells divide uncontrollably and invade body tissues, leading to eventual organ failure and death. These abnormal cells are able to grow and spread by evading recognition and destruction by the immune system through multiple mechanisms. Utilization of immune checkpoints, which are regulators of the immune system that prevent autoimmunity, is one such mechanism exploited by cancer cells. A specific immune checkpoint pathway involves tumor cells upregulating PD-L1 or programmed death ligand 1. When bound to its receptor PD-1, or programmed death protein 1, PD-L1 prevents the anti-tumor immune response normally responsible for clearing abnormal cells throughout the body. Within the past decade, a new class of drugs called immune checkpoint inhibitors have been developed to block this interaction and unleash the patient’s immune system on their tumor cells. While these drugs have seen promising results for some patients, there are still limitations that need to be overcome. Many of the existing cancer immunotherapies are in monoclonal antibody form and have problems with specificity, tissue penetration, route of administration, and are expensive to produce. In this study, we characterized and tested eight different small peptide inhibitors to the PD-1 PD-L1 pathway that were developed using protein painting, a novel technique used to identify specific hotspots within the binding interface. These small peptide inhibitors have the potential to be more specific than antibody therapies by exclusively targeting the protein-protein interface and could potentially be optimized to allow for oral administration. We also characterized various molecular dyes for future protein painting and introduced preliminary studies which optimize cell painting, a novel technique that uses molecular dyes on the surface of living cells to elucidate new drug targets and create whole cell “interactomes” between immune and target cell types.Item Investigation of Urinary Peptides in Congenitally Infected Chagas Disease PatientsAlmofeez, Raghad; Almofeez, Raghad; Luchini, AlessandraChagas disease (CD), caused by the protozoan Trypanosoma cruzi, is mainly transmitted through vectors, blood transfusions or from mothers to their infants. It is estimated that around 25% of new CD infections occur through congenital transmission. In most CD cases, the infection remains asymptomatic for years; however, in an estimated 20-30% of untreated patients, it can cause severe cardiac or gastrointestinal complications, or even death. Detecting and treating CD in the asymptomatic phase is critical as it allows for more efficient treatment before tissue damage progresses to an irreversible stage. CD is endemic in parts of Latin America and due to immigration, it's been growing to be a global public health concern. Current tests use blood specimens for diagnostics; most lack sensitivity and a combination of two independent tests is required. Our study presents a novel non-invasive nanoparticle-enhanced mass spectrometry method to detect T. cruzi peptides in the urine of infants with Chagas disease. Nanoparticles harvest and concentrate low abundance protein markers, preventing their degradation while excluding high molecular weight, abundant proteins. 193 T. cruzi derived peptides were identified in 15 congenitally infected Chagas infants living in endemic areas. Of the identified peptides, mucin-associated surface protein, trans-sialidase and dispersed gene family protein 1 (DGF-1) were highly represented in the urinary peptidome of Chagas patients. Members of those family groups are involved in host invasion and pathogenesis. In this study, we demonstrate for the first time that the nanoparticle-enhanced mass spectrometry test is able to detect urinary T. cruzi peptides in patients with Chagas disease. Few of those peptides were validated using two orthogonal technologies: Parallel reaction monitoring and Western Blot. Introducing a low cost and non-invasive test can be valuable for universal screening of Chagas disease for immigrants and for increasing endemic areas diagnostic testing.Item Nanotechnology Enhanced Immunoassay for Preeclampsia(2014-03-09) Zaidi, Syeda Fatima A.; Zaidi, Syeda Fatima A.; Luchini, AlessandraN-isopropylacrylamide (NIPAm)-based, bait-loaded hydrogel particles are successfully used to capture, sequester and protect from enzymatic degradation low abundance, low molecular weight labile proteins in body fluids. In current protocols, proteins captured by the nanoparticles are eluted with chemical buffers and analyzed with immunoassays or mass spectrometry techniques. Here, a novel, in situ ELISA is presented in which captured proteins are probed with a labeling antibody directly inside the nanoparticles without the need of chemical elution. Dually crosslinked (degradable N,N’-(1,2-Dih ydroxyethylene)- bisacrylamide [DHEA] and nondegradable N,N′-methylenebis(acrylamide) BIS] crosslinkers) NIPAm particles functionalized with trypan blue were used. DHEA can be degraded by an oxidizing agent (e.g. NaIO4) thus increasing the pore size of the hydrogel nanoparticles and allowing the antibody to access the antigen previously trapped in the nanoparticle. Interleukin 6 (IL6) was chosen as a model molecule to test the nanoparticle based ELISA. IL-6 is a protein associated with preeclampsia, an autoimmune, hypertensive pregnancy-related medical problem that can lead to serious complications that can be life threatening to mother and child. Dually crosslinked NIPAm particles efficiently sequestered IL6 from human urine. Oxidative degradation of DHEA crosslinker by NaIO4, caused a partial erosion of the DHEA crosslinker resulting in loose network of hydrogel polymer and increased access of antibody to the captured IL6 protein. Captured IL6 was preserved from oxidative degradation by virtue of the interaction between the protein and trypan blue chemical bait. The IL6 ELISA, based on these dually cross linked NIPAm particles, showed a limit of detection of 600 picograms and two orders of magnitude dynamic range. Coefficient of variations was constantly below 5%. Sensitivity of the test can be improved by increasing the volume of urine processed. It is anticipated that this technology can be successfully used to detect true positive preeclamptic patients from a group of gestational hypertensive population.Item Nanotechnology-enhanced Blood and Urine analysis for the identification of biomarkers related to Severe Traumatic Brain Injury and Acute Respiratory Distress SyndromeVuong, Ngoc; Vuong, Ngoc; Luchini, AlessandraTraumatic brain injuries (TBIs) are physical damages to cerebrum tissue, resulting in temporary or permanent debilitation of brain function. Each year, TBIs contribute to 135,000 deaths and cases of permanent disability in the United States. Acute Respiratory Distress Syndrome (ARDS) is a life-threatening lung condition, which is usually identified by symptoms, including dyspnea, severe hypoxemia, decreased lung compliance, and diffuse bilateral pulmonary infiltrates. ARDS occurs in patients who are significantly ill or are hospitalized due to severe injuries, one of which is TBI. Researchers have been on a quest to find biomarkers for brain injury and its complications in different biofluids. Still, clinically validated TBI biomarkers in urine and serum are lacking sensitivity and specificity. In this study, we applied an affinity nanotechnology and mass spectrometry to discover biomarker candidates related to TBI and ARDS in urine. We analyzed 75 samples (52 = TBI patients, 10 = ARDS patients and 13 = Controls including patients that underwent trauma but did not develop TBI or ARDS). 8 biomarkers related to TBI and 4 biomarkers related to ARDS were selected using Receiver Operating Characteristic analysis. Candidate biomarkers were related to the following biological functions: acute inflammation, cell death, anti-oxidation, endothelial cell repair and regeneration, pulmonary fibrosis and amyloid-beta plaque formation. TBI biomarkers detected in urine are compared with serum TBI biomarkers observed in the preliminary study. Comparison of biomarkers identified in urine and serum revealed that urine yielded a higher number of TBI and ADRS candidate biomarkers (4 and 8, respectively)Item Targeting Rab6/Km23-1 Mediated Compartmentalized Trafficking as a Novel Therapeutic Approach to Alzheimer's DiseaseZhang, Ruth; Zhang, Ruth; Luchini, AlessandraAlzheimer’s Disease (AD) is a prevalent neurodegenerative disease in not only the US but worldwide. Approximately, as of 2020, around 5.8 million Americans, ages 65 and over may be living with AD. AD is a disease where the patient slowly has their mental process deteriorate, causing the patient to not be able to perform everyday tasks, causing an amnesia-like memory retention, and eventually, in severe cases, ending in an vegetative state, and requiring 24/7 care. AD can either be passed down generation to generation or cases can be sporadic, which is more common. Through the years, researchers have focused on an array of hypotheses on the disease pathogenesis and the Amyloid Hypothesis is the most studied. At the molecular level, amyloid beta (Aβ) plaques derive from the breakdown of a protein names amyloid precursor protein (APP). Amyloid fragments clump together and form plaques that accumulate between neurons and destroy cell functions. Many therapies have been targeted to the downstream effects of amyloid beta formation. Unfortunately, these therapies had little success in reducing the cell brain destruction caused by AD so a novel and fresh approach to AD therapy is greatly needed. In this study, we focus on two proteins, Rab6 and Km23-1 that may play an important role in the formation of amyloid plaques by mediating trafficking of APP in the neuronal cell. Our hypothesis is that the interaction between Km23-1 and Rab6 contributes to the cleavage of APP through over reactive Golgi trafficking, which leads to Aβ plaque formation. If this hypothesis is confirmed, targeting the Rab6/Km23-1 protein complex becomes a potentially attractive pharmacological strategy for AD. The work presented in this thesis contributes two major achievements: for the first time, we demonstrated that Rab6 and Km23-1 form a protein complex in vitro, and 2) we developed an experimental pipeline to use the mass spectrometry based protein painting technology to identify functionally relevant hot spots of interaction. Information derived from protein painting can be used to design specific and potent inhibitors to disrupt the Rab6/Km23-1 protein complex.Item The Characterization of Tuberculosis Urinary Peptidome in Pediatric PatientsAlmosuli, Mahmood; Almosuli, Mahmood; Luchini, AlessandraTuberculosis is considered as the primary source of death caused by infectious diseases globally. Millions of people have been either infected or killed by tuberculosis in the year of 2018 alone. Countries that have health and economic challenges have high mortality rates of tuberculosis in children. Moreover, children infected with TB represent the main reservoir for future cases. Early diagnosis of the children can increase treatment efficacy and prevent future spread. The conventional diagnostic methods for TB are not very effective in diagnosing children due to the paucibacillary nature of TB, and the inability of children to produce sputum. Urine has the potential to be used as a biofluid for the identification of TB because it can be easily collected non-invasively. TB urine testing have been impeded in the past due to the low concertation of TB antigens that degrade rapidly, and the presence of highly abundant proteins in urine. The development of a novel nanoparticle biomarker harvesting technology has facilitated the use of urine for TB testing. In this study, we will attempt to discover novel Mycobacterium tuberculosis derived markers in the urine of pediatric patients using novel hydrogel nanocage affinity bait biomarker harvesting technology.