The role of megakaryocytes and platelets in infection and immunothrombosis

Thesis: Sc. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2018.Cataloged from PDF version of thesis. Includes CD-ROM with 9 videos in the .avi format and 2 video in the .mp4 format.Includes bibliographical references.Megakaryocytes (MKs), one of the largest and rarest hematopoietic stem cells in the bone marrow, have traditionally played a primary role in hemostasis as precursors to platelets, which are importantly, one of the most abundant cell types in the peripheral circulation. While platelets are studied for their various roles in inflammation, the role of MKs within the innate immune system has not been explored. In a series of comprehensive in vitro experiments, we have demonstrated that both cord blood-derived MKs and MKs from a megakaryoblastic lineage have innate immune cell functions, including: phagocytosis, formation of extracellular traps, and chemotaxis towards pathogenic stimuli. MKs were also observed to directionally release platelets towards pathogenic stimuli. In addition to their primary role as immune cells, MKs were also shown to contain extranuclear histones, which the MKs release along with budding platelets into the circulation. These small packages of histones can play a major role in inflammation and immunothrombosis by promoting inflammation and coagulation. By evaluating blood and tissue samples from patients diagnosed with sepsis, we demonstrated that there is an increased MK concentration both in the peripheral circulation, as well as in the lungs and kidneys. Platelets from patients with sepsis also appeared to have a specific phenotype, including increased DNA and histone staining. MK number in the circulation and end-organs, as well as platelet histone expression appeared to be correlated with both prognosis and type of infection. This newly recognized role of MKs as functional innate immune cells may have significant implications for the role of MKs in conditions such as sepsis and, pending a more profound mechanistic understanding, may further lead to the development of novel targets for the treatment of sepsis.by Galit Hocsman Frydman.Sc. D

Helicobacter pylori Eradication in Patients with Immune Thrombocytopenic Purpura: A Review and the Role of Biogeography

Idiopathic thrombocytopenic purpura (ITP) is typically a diagnosis of exclusion, assigned by clinicians after ruling out other identifiable etiologies. Since a report by Gasbarrini et al. in 1998, an accumulating body of evidence has proposed a pathophysiological link between ITP and chronic Helicobacter pylori (H. pylori) infection. Clinical reports have described a spontaneous resolution of ITP symptoms in about 50% of chronic ITP patients following empirical treatment of H. pylori infection, but response appears to be geography dependent. Studies have also documented that ITP patients in East Asian countries are more likely to express positive antibody titers against H. pylori-specific cytotoxic-associated gene A (CagA), a virulence factor that is associated with an increased risk for gastric diseases including carcinoma. While a definitive mechanism by which H. pylori may induce thrombocytopenia remains elusive, proposed pathways include molecular mimicry of CagA by host autoantibodies against platelet surface glycoproteins, as well as perturbations in the phagocytic activity of monocytes. Traditional treatments of ITP have been largely empirical, involving the use of immunosuppressive agents and immunoglobulin therapy. However, based on the findings of clinical reports emerging over the past 20 years, health organizations around the world increasingly suggest the detection and eradication of H. pylori as a treatment for ITP. Elucidating the exact molecular mechanisms of platelet activation in H. pylori-positive ITP patients, while considering biogeographical differences in response rates, could offer insight into how best to use clinical H. pylori eradication to treat ITP, but will require well-designed studies to confirm the suggested causative relationship between bacterial infection and an autoimmune disease state.National Institutes of Health (U.S.) (T320D010978-26)National Institutes of Health (U.S.) (P01CA028842-23)National Institutes of Health (U.S.) (P30ES002109

The E2 Ubiquitin-conjugating Enzyme UBE2J1 Is Required for Spermiogenesis in Mice

ER-resident proteins destined for degradation are dislocated into the cytosol by components of the ER quality control machinery for proteasomal degradation. Dislocation substrates are ubiquitylated in the cytosol by E2 ubiquitin-conjugating/E3 ligase complexes. UBE2J1 is one of the well-characterized E2 enzymes that participate in this process. However, the physiological function of Ube2j1 is poorly defined. We find that Ube2j1−/− mice have reduced viability and fail to thrive early after birth. Male Ube2j1−/− mice are sterile due to a defect in late spermatogenesis. Ultrastructural analysis shows that removal of the cytoplasm is incomplete in Ube2j1−/− elongating spermatids, compromising the release of mature elongate spermatids into the lumen of the seminiferous tubule. Our findings identify an essential function for the ubiquitin-proteasome-system in spermiogenesis and define a novel, non-redundant physiological function for the dislocation step of ER quality control.United States. National Institutes of Health (P30-CA14051

Ataxia-telangiectasia: Linkage analysis in highly inbred Arab and Druze families and differentiation from an ataxia-microcephaly-cataract syndrome

Ataxia-telangiectasia (A-T) is a progressive autosomal recessive disease featuring neurodegeneration, immunodeficiency, chromosomal instability, radiation sensitivity and a highly increased proneness to cancer. A-T is ethnically widespread and genetically heterogeneous, as indicated by the existence of four complementation groups in this disease. Several "A-T-like" genetic diseases share various clinical and cellular characteristics with A-T. By using linkage analysis to study North American and Turkish A-O families, the ATA (A-T, complementation group A) gene has been mapped to chromosome 11q23. A number of Israeli Arab A-T patients coming from large, highly inbred families were assigned to group A In one of these families, an additional autosomal recessive disease was identified, characterized by ataxia, hypotonia, microcephaly and bilateral congenital cataracts. In two patients with this syndrome, normal levels of serum immunoglobulins and alpha-fetoprotein, chromosomal stability in peripheral blood lymphocytes and skin fibroblasts, and normal cellular response to treatments with X-rays and the radiomimetic drug neocarzinostatin indicated that this disease does not share, with A-T, any additional features other than ataxia. These tests also showed that another patient in this family, who is also mentally retarded, is affected with both disorders. This conclusion was further supported by linkage analysis with 11q23 markers. Lod scores between A-O and these markers, cumulated over three large Arab families, were significant and confirmed the localization of the ATA gene to aq23. However, another Druze family unassigned to a specific complementation group, showed several recombinants between A-T and the same markers, leaving the localization of the A-T gene in this family open

Predominance of null mutations in ataxia-telangiectasia

Ataxia-telangiectasia (A-T) is an autosomal recessive disorder involving cerebellar degeneration, immunodeficiency, chromosomal instability, radiosensitivity and cancer predisposition. The responsible gene, ATM, was recently identified by positional cloning and found to encode a putative 350 kDa protein with a PI 3-kinase-like domain, presumably involved in mediating cell cycle arrest in response to radiation-induced DNA damage. The nature and location of A-T mutations should provide insight into the function of the ATM protein and the molecular basis of this pleiotropic disease. Of 44 A-T mutations identified by us to date, 39 (89%) are expected to inactivate the ATM protein by truncating it, by abolishing correct initiation or termination of translation, or by deleting large segments. Additional mutations are four smaller in-frame deletions and insertions, and one substitution of a highly conserved amino acid at the PI 3-kinase domain. The emerging profile of mutations causing A-T is thus dominated by those expected to completely inactivate the ATM protein. ATM mutations with milder effects may result in phenotypes related, but not identical, to A-T

The Potential Role of Coagulation Factor Xa in the Pathophysiology of COVID-19: A Role for Anticoagulants as Multimodal Therapeutic Agents

SARS-CoV-2 infection (COVID-19) results in local and systemic activation of inflammation and coagulation. In this review article, we will discuss the potential role of coagulation factor Xa (FXa) in the pathophysiology of COVID-19. FXa, a serine protease, has been shown to play a role in the cleavage of SARS-CoV-1 spike protein (SP), with the inhibition of FXa resulting in the inhibition of viral infectivity. FX is known to be primarily produced in the liver, but it is also expressed by multiple cells types, including alveolar epithelium, cardiac myocytes, and macrophages. Considering that patients with preexisting conditions, including cardiopulmonary disease, are at an increased risk of severe COVID-19, we discuss the potential role of increased levels of FX in these patients, resulting in a potential increased propensity to have a higher infectious rate and viral load, increased activation of coagulation and inflammation, and development of fibrosis. With these observations in mind, we postulate as to the potential therapeutic role of FXa inhibitors as a prophylactic and therapeutic treatment for high-risk patients with COVID-19

Coagulation Status and Venous Thromboembolism Risk in African Americans: A Potential Risk Factor in COVID-19

Severe acute respiratory syndrome coronavirus 2 infection (COVID-19) is known to induce severe inflammation and activation of the coagulation system, resulting in a prothrombotic state. Although inflammatory conditions and organ-specific diseases have been shown to be strong determinants of morbidity and mortality in patients with COVID-19, it is unclear whether preexisting differences in coagulation impact the severity of COVID-19. African Americans have higher rates of COVID-19 infection and disease-related morbidity and mortality. Moreover, African Americans are known to be at a higher risk for thrombotic events due to both biological and socioeconomic factors. In this review, we explore whether differences in baseline coagulation status and medical management of coagulation play an important role in COVID-19 disease severity and contribute to racial disparity trends within COVID-19

Device Prototype for Vaginal Delivery of Extremely Preterm Fetuses in the Breech Presentation

© 2021 by ASME. Vaginal delivery is typically avoided in the extremely preterm breech population due to the concern of entrapment by the cervix of the aftercoming head. A mechanical device concept is presented to enable vaginal delivery by preventing retraction of the cervix against the fetus during delivery. The two-part device was designed to dilate the cervix, prevent prolapse of small fetal parts and maintain sufficient dilation during delivery. The two-part device was designed and manufactured with the following modules: an inflatable saline-filled cervical balloon for dilation and a cervical retractor composed of semirigid beams to stabilize the cervix and maintain adequate dilation. The device was tested using a cervical phantom designed to simulate the compressive force the cervix exerts. The cervical balloon reached a maximum dilation of 8.5 cm, after which there was leakage of saline from the balloon. While this dilation was less than the target goal of 10 cm, the leaking was attributed to prototype manufacturing defects, which could be resolved with further development. The cervical retractor was able to withstand between 1-3 kPa. Although estimates of cervical pressure values can be upward of 30 kPa, there are no in vivo measurements to formally identify the pressure values for patients in preterm labor. This device serves as a viable proof-of-concept for utilizing an inflatable balloon device to prevent cervical retraction in the setting of extremely preterm vaginal breech delivery. Further manufacturing improvements and design changes could improve the device for continued development and testing

Manuka honey microneedles for enhanced wound healing and the prevention and/or treatment of Methicillin-resistant Staphylococcus aureus (MRSA) surgical site infection

© 2020, The Author(s). Manuka honey (MH) is currently used as a wound treatment and suggested to be effective in Methicillin-resistant Staphylococcus aureus (MRSA) elimination. We sought to optimize the synthesis of MH microneedles (MHMs) while maintaining the MH therapeutic effects. MHMs were synthesized using multiple methods and evaluated with in vitro assays. MHMs demonstrated excellent bactericidal activity against MRSA at concentrations ≥ 10% of honey, with vacuum-prepared honey appearing to be the most bactericidal, killing bacterial concentrations as high as 8 × 107 CFU/mL. The wound-healing assay demonstrated that, at concentrations of 0.1%, while the cooked honey had incomplete wound closure, the vacuum-treated honey trended towards faster wound closure. In this study, we demonstrate that the method of MHM synthesis is crucial to maintaining MH properties. We optimized the synthesis of MHMs and demonstrated their potential utility in the treatment of MRSA infections as well as in wound healing. This is the first report of using MH as a substrate for the formation of dissolvable microneedles. This data supports the need for further exploration of this new approach in a wound-healing model and opens the door for the future use of MH as a component of microneedle scaffolds