bluuebunny/crossref_metadata_2025 huggingface dataset:
AMIGO-ENEMIGO EL CAMINO HACIA LA CONSTRUCCIÓN DEL ESTADO EN LA REPÚBLICA DE ARGENTINA. ANÁLISIS DE LA OBRA FACUNDO DE DOMINGO FAUSTINO SARMIENTO
Authors: Cely-Ospina Jovany , Quintero-Ortega Jasney
Published: August 2023

El propósito del siguiente análisis de la obra Facundo escrita por Domingo Faustino Sarmiento, es el de mostrar el camino hacia la construcción del Estado en la República Argentina, en términos de Weber, pero añadiendo que el camino para la construcción de esta institución se caracterizó en una diferenciación entre amigo-enemigo como plantea Schmitt. Diferenciación que permitió, en parte, controlar los medios legítimos de coacción física dentro del territorio, además, de permitir establecer vínculos regionales, los cuales han estado influenciados por la figura del caudillo. Que para la época de Juan Manuel de Rosas fueron de vital importancia, ya que la política de Rosas se basaba única y exclusivamente, como lo deja entrever Sarmiento, en el que no está conmigo, es mi enemigo. Para ello, resulta de importancia, plantear el contexto latinoamericano en que se ubica la obra literaria, seguido, exponer la figura del caudillo con Rosas y, por último, analizar la consolidación del Estado moderno, en la visión dicotómica “amigo-enemigo” de Carl Schmitt.
Sınıf Öğretmenleri Açısından Bilgi İşlemsel Düşünme ve Kodlama Öğretiminin Kapsamının Belirlenmesine Yönelik Bir İnceleme
Authors: CÜCÜ Hatice , DAĞ Funda
Published: August 2023

“Herkes için bilgisayar bilimleri!” söylemiyle birlikte önce kodlama ve ardından bilgi işlemsel düşünme son yıllarda artan bir ivmeyle Dünya’da birçok ülkede ve Türkiye’de ilgi çeken konular haline gelmiştir. Birçok ülkenin öğretim programlarında kodlama ve bilgi işlemsel düşünme yer almaya başlamıştır. Türkiye’de 2018-19 öğretim yılı itibariyle, kodlama ve bilgi işlemsel düşünmeyi içeren, ilkokul 1-4. sınıf düzeyinde Bilişim Teknolojileri ve Yazılım dersi, serbest etkinlik dersleri kapsamında sınıf öğretmenlerinin kullanımına sunulmuştur. Türkiye’de kodlama öğretimine en çok ilgi gösterenler arasında sınıf öğretmenleri önde gelmektedir. Bu araştırmada; sınıf öğretmenleri açısından bilgi işlemsel düşünme ve kodlama konularına yönelik bir kapsam belirleme incelemesi amaçlanmıştır. Araştırmanın bulgularına göre; sınıf öğretmenlerinin Bilişim Teknolojileri ve Yazılım dersinde bilgi işlemsel düşünme ve kodlamanın yanı sıra, verdikleri tüm derslerde bilişim teknolojileriyle ilgili rol ve sorumluluklarının olduğu belirlenmiştir. Kodlama öğretimi ve bilgi işlemsel düşünme konusunda sınıf öğretmenleri için öğretim alanlarına yönelik eğitim olanaklarının ve öğretim materyali kaynaklarının sınırlı olduğu görülmüştür. Ulusal alanyazında bilgi işlemsel düşünme ve kodlamayla ilgili ilkokul düzeyinde veya sınıf öğretmenlerini konu alan araştırmaların çok az sayıda olduğu belirlenmiştir. Sınıf öğretmenleri açısından bilgi işlemsel düşünme ve kodlama konularında belirsizliklerin giderilmesine yönelik araştırma önerilerine yer verilmiştir.
Producción radiofónica de radio integración aplicada a la educación y desarrollo social en jóvenes del cantón Pindal
Authors: Sandoya Guzmán María Victoria , González Carrión Erika Lucía , Sandoya Valdiviezo César Teodomiro , Aponte Gómez Anny Geomayra , Moreno Loaiza Ulda Maritza , Torres Jiménez Eugenia Maricela
Published: August 2023

Esta investigación constituyó un análisis de la producción radiofónica que la emisora Radio Dinamik orientada a la educación y el desarrollo social de jóvenes en el cantón Pindal, provincia Loja. Se seleccionó esta emisora por ser de las pioneras en expandir su cobertura y una de las más escuchadas en esta zona geográfica de la región y, pese a carecer de la categoría de comunitaria, brinda amplia apertura a la ciudadanía. El estudio empleó una metodología con enfoque mixto con un diseño cuali-cuantitativo. Se aplicaron los métodos inductivo-deductivo, análisis-síntesis. Además, se emplearon las técnicas de entrevista, encuesta, focus group y observación; así como los instrumentos denominados fichas y guías de entrevista y de observación. Se aplicaron alrededor de 150 encuestas aleatorias dirigidas a ciudadanos con edades que oscilan entre 18 a 30 años de edad, para determinar la preferencia de los jóvenes con el tipo de programación que mantiene la emisora en referencia. Así mismo, se entrevistaron a expertos en radio para, junto a la observación, recoger información y establecer en qué medida la programación de la radiodifusora contribuye a la educación y en consecuencia, al desarrollo social. Los resultados obtenidos confirman la necesidad de potenciar en la parrilla de programación segmentos de corte educativo donde los jóvenes se sientan incluidos e identificados. Se concluye que esta emisora apuesta por programas de entretenimiento, informativos y de opinión, en detrimento de programas educativos que permitan una mejor convivencia y un mejor desarrollo social.
Multicultural Education in Indonesian Diaspora Families (Study Case: Gelin Community)
Authors: Anggraeni Nur Najmi , Ustaadza Laillatus Sholikhah , Komalasari Komalasari
Published: August 2023

This writing discusses multicultural education in Indonesian diaspora families in Türkiye. It aims to reveal the application of multicultural education in the Gelin community in Turkey as an attempt to present the identity as Indonesian and measure the understanding of Nationalism and tolerance in the families of the Gelincommunity in Turkey. This writing method uses qualitative research methods with sources Indonesian gelinwho are married to Turks and living in Turkey. The ups and downs of the application of multicultural education are passed in mixed marriages, gelins in Turkey have their own pattern regarding the application of multicultural educaion which they teach to their children. Although there are no official rules for the use of multicultural education, multicultural education is a way to help children from mixed marriages find their identities without being forced to side with two different nationalities.
Evolution and New Generation of Dual Mobility Cups
Authors: Tigani Domenico , Banci Lorenzo , Stallone Stefano , Melucci Giuseppe , Pieratelli Giulio , Castiello Emanuela
Published: August 2023

Although total hip arthroplasty (THA) is considered a successful procedure, hip dislocation remains the main cause of early failure. Dual mobility cups (DMCs) have been shown to significantly reduce the dislocation rate in both primary and revision THAs. During the past several decades, DMCs have evolved in design and fixation interface. There have been three generations of DMCs. This article addresses the rationale for a new cementless highly porous titanium DMC to improve component fixation and implant biocompatibility. [Orthopedics. 2023;46(5):e273–e280.]
Update on Current Concepts of Blood Flow Restriction in the Perioperative Period of Anterior Cruciate Ligament Reconstruction
Authors: Hasegawa Morgan E. , Delos Reyes Chloe D. , Rimm Julian B. , Radi Joshua K. , Singh Dylan S. , Obana Kyle K. , Weldon Edward J. , Thorne Tyler J. , Tamate Trent M. , Alferos Sarah Rosabelle , Min Kyong S.
Published: August 2023

Anterior cruciate ligament tears or ruptures are common orthopedic injuries. Anterior cruciate ligament reconstruction (ACLR) is an orthopedic procedure allowing for earlier return to sports, improved maintenance of lifestyle demands, and restored knee stability and kinematics. A perioperative rehabilitative adjunct recently gaining interest is blood flow restriction (BFR), a method in which temporary restriction of blood flow to a chosen extremity is introduced and can be used as early as a few days postoperative. There has been increasing investigation and recent literature regarding BFR. This review synthesizes current concepts of BFR use in the ACLR perioperative period. [Orthopedics. 2023;46(6):e333–e340.]
Factors affecting the transmission of dengue fever in Haikou city in 2019
Authors: Zheng Zuohuan , Wang Kaihua , Yang Daiyu , Yin Feifei , Sun Dingwei , Yu Weiyan , Lin Jialun , Liu Ying , Chen Changkuan , Yang Zehui , Wu Biao
Published: August 2023

In this study, due to multiple cases of dengue fever in two locations in Haikou, Hainan, several factors affecting the transmission of dengue fever in Haikou in 2019 were analyzed. It was found that dengue fever spread from two sites: a construction site, which was an epidemic site in Haikou, and the university, where only four confirmed cases were reported. Comparative analysis revealed that the important factors affecting the spread of dengue fever in Haikou were environmental hygiene status, knowledge popularization of dengue fever, educational background, medical insurance coverage and free treatment policy knowledge and active response by the government.
Materialistična teorija sreče
Authors: Nedoh Boštjan
Published: August 2023

Če bi iskali kandidata za najbolj kontroverzen pojem danes, bi »sreča« nedvomno bila med favoriti za zasedbo tega mesta. Po eni strani je sreča v zgodovini filozofije predstavljala eno osrednjih etičnih torišč – od Grške eudaimonie do Benthamovega imperativa »največja sreča za največje število ljudi«. Etika je bila skoraj neločljivo povezana z iskanjem sreče, ki je že pri Grkih delovala kot sinonim »dobrega življenja«. Po drugi strani kapitalizem danes, kljub splošni apatiji in brezupnosti ljudi zaradi permanentnih kriz kapitalističnega sistema (podnebne spremembe, pandemija, finančna kriza itd.), samega sebe še vedno v precejšnji meri legitimira, če že na z zagotovilom, pa vsaj z obljubo sreče. Navkljub različnim zgodovinskim kontekstom, iz katerih je pojem sreče vedno znova dobival svoj pomen, pa vendarle lahko med vsemi temi razlikami izluščimo nek skupni imenovalec, ki je danes, v dobi poznega kapitalizma, celo pridobil status temeljnega kamna naše hegemone predstave o sreči. Sreča zadeva vprašanje biti. Ob evokaciji sreče se običajno poraja vprašanje, ali je nekdo srečen in kaj pomeni biti srečen oziroma kako to postanemo. Sreča je tako neločljivo povezana z vprašanjem identitete: je tako rekoč čutni izraz kvalitet naše biti, tega, kar domnevno smo. V nasprotju s to »ontologizacijo sreče« si članek prizadeva pokazati ne samo, da obstaja materialistični pojem sreče, temveč tudi, da ta pojem temelji na primatu glagola »imeti« nad glagolom »biti«. Z materialistične perspektive je sreča nekaj, kar lahko imamo oziroma nas doleti, ne pa nekaj, kar smo. Pri tem sta ključni dve referenci: prvič, Benjaminova formulacija sreče iz njegovega zadnjega teksta »O pojmu zgodovine«, in, drugič, freudovsko-lacanovska teorija ponavljanja.
Sensation(all) Ontology
Authors: Zeiher Cindy
Published: August 2023

It is the praxis of being a subject in the world which enables psychoanalysis to theorise subjectivity. Freud theorised subjectivity from the perspective of desires, those repressed unconscious forces which conflict with the subjects’ need to live in the world. The upshot of this conflict for the subject is trauma and for psychoanalysis such trauma provides a way into a remedy, a cure, the presumption of psychoanalysis being that through its method of transference, it does indeed possess the knowledge to pursue a remedy. Lacan offers a new interpretation of Freud by considering the subject as an ongoing ontological enigma in so far as subjective unconscious desire is not only (potentially) traumatic but necessarily (always) linguistic. How is this an ontological enigma? For Lacan the subject is first and foremost a speaking being engaged in an ongoing struggle to articulate unconscious desire. This is because, claims Lacan, we are born into language which not only pre-exists us but continues after death. In this way subjectivity is inescapably oriented to language as simultaneously intrinsic external to it. The unconscious is therefore a mixture of inside and outside, an enigmatic (pre)ontological space which Lacan calls extimacy. This essay seeks to explore Lacan’s orientation of the subject towards extimacy as the site of subjective conflict where, in its quest for the subject’s desire to know and handle the symptom, transference engages a new ontological dimension in which we can say that the extimate is structured like a sensation.
Fresh, hardened and durability properties of sodium carbonate-activated Algerian slag exposed to sulfate and acid attacks
Authors: Kahlouche R. , Badaoui A. , Criado M.
Published: August 2023

This paper investigates the use of Na2CO3 as an alkaline activator on the durability of the alkali-activated slag (AAS) mortar toward sulfates and acids. The behavior of this binder in these aggressive environments is compared to those of slags activated with Na2SiO3 and NaOH. In addition, the setting times, workabilities, mechanical properties and drying shrinkage were evaluated. The AAS had superior workabilities, faster setting times and higher shrinkage rates than the Portland cement (PC). Increases in the activator dosages had positive effects on the mechanical strengths of the materials. Na2SiO3 was the best activator in terms of strength development, but it led to much higher shrinkage. The AAS showed less expansion and lower weight losses than the PC when exposed to sulfate and acids, respectively. The Na2CO3-AAS exhibited less shrinkage and higher resistance to sulfuric acid than the other activators, but the mechanical strength seen at early ages was low.
Experimental investigation of clogging mechanism of pervious concrete made with variable aggregate gradations
Authors: Nazeer M. , Kapoor K. , Singh S.P.
Published: August 2023

In this study, the clogging mechanism of pervious concrete was evaluated using three different cloggers such as, Sand (S), Clay (C), and combination of sand and clay (S &C). The clogging mechanism was performed through falling head permeability apparatus, using clogger sediment load at the rate of 50, 150, and 200 grams in repetitive clogging cycles. It was observed from the results that combined (S & C) clogger shows overall critical results of clogging as 80% of the clogging was seen in 3 to 4 cycles. Moreover, it was observed from the results that pervious concrete mix made with R- type of aggregate gradation shows optimum compressive strength of the order of 8.6, 15.9, and 17 MPa at 7, 28 and 56 days of curing. Furthermore, the visual inspection test shows that clogging by clay clogger shows an even distribution of sediment on the whole length of the sample.
AML/CFT Regulations and Informal Remittance Services
Authors: Jayasekara S. G. Sisira Dharmasri , Rafay Abdul
Published: August 2023

Informal remittance channels operate in various jurisdictions to promote illegal activities which are detrimental to the global efforts of combating money laundering. Here, in this chapter, Hawala is considered the main informal remittance channel. The economic and social implications of Hawala are very significant when it comes to a weak regime of AML/CFT. Therefore, the focus of this chapter is on weak AML/CFT regimes even though the discussed practices take place across the globe. A global initiative to limit or control informal remittance channels may support the global initiatives of anti-money laundering. However, it has also to bear in mind that informal remittance services are used globally as a very efficient and low-cost remittance channel despite the allegations of using them by criminals. Therefore, it is very important to implement proper systems and controls to regulate informal remittance channels while making them available for all segments of society.
The Effect of Corruption Perception on Foreign Direct Investment
Authors: Ferreira Thiago , Lirio Viviani Silva , Clemente Felippe , Oke Tayo
Published: August 2023

Economists have been studying the effects of corruption on a variety of economic variables, like growth, inequality and innovation. This chapter investigates the impact of Transparency International's Corruption Perception Index (CPI) on the inflows of Foreign Direct Investment to Latin American Countries for the period 2012-2019. The choice of the period is due to the new methodology of the CPI which allows for comparisons between time periods. A panel data model is employed to investigate whether corruption “grease the wheels” or “sand the wheels” of FDI. Results show that the CPI exerts no influence on the inflows of FDI, at least for this time period and sample. The chapter also considered the criticism raised by Heywood (2017), who criticizes the very use of measures of perception of corruption as valid parameters to analyze the role of corruption on economic outcomes.
Environmental Non-Governmental Organizations' Tax Regime
Authors: Rocha Ana Paula
Published: August 2023

In the most recent years, the Environmental Non-Governmental Organizations (commonly known as Environmental NGO's) had assumed a very important role in the defense and enhancement of the environment and natural heritage. In this sense, the tax regime applicable to those entities can be framed as a green tax regime, to the extent that it may constitute an instrument for the State' intervention towards the protection of the environment. Therefore, in this chapter, the author addresses the main topics concerning the Portuguese tax regime applicable to the Environmental NGO's, not only in what concerns to the framework specifically applicable to those organizations under the main Portuguese' taxes but, also, in what concerns to the tax benefits that, in respect for the fundamental principle of tax justice, can be recognized to them.
Parents' View of Graphic Novels in Hong Kong Under the 21st Century Mobile Digital Environment
Authors: Chan Samuel Kin Fung , Chan Mimi Mei Wa , Lam Apple Hiu Ching , Chiu Dickson K. W.
Published: August 2023

This study reviews the current parents' perspectives and habits of using graphic novels, including electronic equipment, in Hong Kong to support children's reading and learning. Parents were invited to participate in semi-structured group interviews based on our five research questions. The authors employ a thematic analysis approach to analyze the data and determine the similarities and differences between the literature review and the current mobile digital environment. The findings indicated that respondents were not keen on using graphic novels to support their children's reading and learning. The two main reasons were inadequate understanding or bias on graphic novels and the wide availability of online digital materials for reading and learning support. Scant studies focus on graphic novels in parent-child reading from parents' perspectives, especially in the Asian context, though graphic novels are becoming a worldwide trend. Parents' opinions about this issue are invaluable for educators and librarians in curriculum design and collections development.
Accessing User Satisfaction of Makerspace in Academic Libraries
Authors: Cheung Cho Yiu , Lam Apple Hiu Ching , Chiu Dickson K. W.
Published: August 2023

Makerspaces have developed from a trend to a core service in higher education libraries. Many academic libraries have been actively expanding the “makerspace” within the physical library and revitalizing the library as a center of learning and innovation. This case study investigates the application of makerspace technologies in a major comprehensive library in Hong Kong, which has designed a specific makerspace to encourage innovation and creativity. Few studies have focused on in-depth studies of makerspaces in East Asian academic libraries and how patrons perceive makerspace services and innovative spaces. A survey instrument was developed using the 5E instructional model (engage, explore, explain, elaborate, and evaluate) to evaluate makers' experiences systematically. The finding revealed that respondents applauded the importance of innovative spaces and the perceived outcomes from makerspaces, including nurturing creativity and critical thinking. However, they did not have sufficient skills to use emerging technologies, resulting in low usage.
Discovery and characterization of cross-reactive single-domain antibodies targeting key virulence factors of Salmonella and Shigella
Authors: Ochoa Mark Anthony , Schmidt Hayden R
Published: August 2023

Study Objective

Here, we utilize a nanobody yeast display library to identify nanobodies that target virulence factors in Shigella spp. and Salmonella enterica typhimurium and determine if these nanobodies (Nbs) can inhibit host cell invasion in vitro.

Brief Statement of Methods

Using a synthetic yeast display library containing camelid Nbs with randomized CDR loops, we selected for binders to select Enterobacteriaceae virulence proteins, including IpaD/SipD, IcsA, and IcsP by MACS and FACS. Lead Nbs were cloned and expressed as monomeric VHH domains or Nb-Fc fusions. ELISA and flow cytometry surface staining was used to estimate affinity, and functional activity was assayed via a host cell invasion assay with Shigella flexneri, Shigella sonnei, and Salmonella enterica typhimurium.

Summary of Results

Several high affinity Nbs were isolated with specificity for the targe proteins, and a subset of these could bind whole bacterial cells. Lead Nbs were binned into epitopes by competitive binding assays. The efficacy of Nbs against specific epitopes of specific virulence proteins in invasion assays were directly compared.

Statement of Conclusions

A direct comparison of high-affinity Nb binders against various epitopes of IpaD/SipD, IcsA, and IcsP reveals that only a subset of epitopes are accessible on whole bacteria and able to inhibit bacterial invasion. These results will inform the development of virulence factor-based vaccines or other immunotherapies.

Wellcome Trust (REISTANT Consortium)
Fishing for novel dengue broadly neutralizing antibodies
Authors: Lowman Kelsey Elizabeth , Johnston Tim , Aogo Rosemary A , Thiono Devina , Mpingabo Patrick , Henry Amy R , Robert-Torres Jesmine , Estrada Luis , Lima Noemia Santana , de Silva Aravinda M , Douek Daniel , Katzelnick Leah
Published: August 2023

Due to a patent filing, the content of this abstract is not available to be shared at this time. The abstract will be shared at the poster session.

This work was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health.
Development of a potent and infection-resistant anti-HIV CAR-T cells therapy
Authors: Lou Yaoxian , He Xiuting , Li Guangming , Su Lishan
Published: August 2023

Acquired immune deficiency syndrome (AIDS) caused by HIV infection is a serious global public health issue. The current combined antiretroviral therapy (ART) effectively controls HIV replication but fails to eliminate HIV reservoir cells, which allows a persistent viral infection. HIV-specific chimeric antigen receptor (CAR) T-cells have been designed to eliminate HIV-infected cells. The broad anti-viral activity, resistance to HIV infection and immune suppressive microenvironment determine a successful CAR-T therapy for HIV infection. In this study, we developed a potent CD4-based CAR-T cell with herpes virus entry mediator (HVEM) costimulatory domain. We intruded a S60C mutation in CD4 extracellular domain, which mediates a higher binding affinity to HIV envelope glycoprotein gp120 while preventes gp41-induced fusion between viral envelope and cell membrane. We demonstrated that CD4-based CAR-T cells bearing this 2C mutation were resistant to HIV infection. In addition, the HVEM costimulatory domain (CSD) mediated a superior effector T cell function than CD28 or 4-1BB-derived CSD did in CAR-T cells, showed by either cytotoxicity effect or cytokines response in contact with the HIV Envelope-transduced K562 cells in vitro. Furthermore, HVEM-CAR-T showed a better therapeutic efficacy than CD28 or 4-1BB CAR-T in humanized mouse model in vivo. Overall, we designed and developed a potent infection-resistant anti-HIV CAR-T cell by incorporating a mutant CD4 extracellular domain and HVEM-derived CSD.
Chlamydia muridarum recombinant MOMP nanovaccine induces cross-reactive antibodies against Chlamydia trachomatis human serovars
Authors: Sahu Rajnish , Sipowe Aguy Clemence Nguiakam , Tadjuidje Vanella , Geisler William M , Van Der Pol Barbara , Dennis Vida A
Published: August 2023

Chlamydia trachomatis (CT), caused by various serovars, remains the leading sexually transmitted bacterial infection worldwide. An efficient delivery system is critical to developing whole or subunit vaccines against CT, and some vaccine developmental efforts are targeting biodegradable nanoparticles-based vaccines with encapsulated antigens. We developed a nanovaccine employing the recombinant major outer membrane protein (rMOMP) of C. muridarum (Cm) encapsulated in poly(lactic acid-co-glycolic acid) 85/15 (PLGA-rMOMP). We previously compared the humoral responses of two priming routes, subcutaneous (SC) or intramuscular (IM-p), followed by two SC-boosts to evaluate rMOMP-specific serum antibodies [IgG2a, IgG2b and IgG1] produced by PLGA-rMOMP immunization of female BALB/c mice. Here we used an elementary body (EB)-specific ELISA to investigate the ability of systemic antibodies produced in immunized mice to recognize Cm and to cross-recognize human CT-serovars D, F and J. Serum from mice inoculated with PBS encapsulated PLGA served as negative control. We observed high recognition of Cm specific total IgG (8-fold) and isotypes IgG2a (8-fold), IgG2b (32-fold), IgG1 (8-fold) by SC in comparison to IM-p immunizations. We also observed IgG antibodies recognizing CT-serovars D, F and J from mice immunized via both routes. Evaluation of the Th1 (IgG2a, IgG2b)/Th2 (IgG1) antibody titer ratios revealed that immunization via the SC route induced predominantly Th1 antibodies recognizing both Cm and human CT-serovars. Our data show that mice immunized with PLGA-rMOMP produced high levels of systemic antibodies recognizing Cm, but more importantly, there was a robust serological cross-recognition of the human CT-serovars.

This research was supported by the National Institute Of Allergy And Infectious Diseases of the National Institutes of Health under Award Number NIH-R21AI111159, NIH-NIGMS-RISE (1R25GM106995-01) and the National Science Foundation (NSF)-CREST (HRD-1241701) and NSF-HBCU-RISE (HRD-1646729) grants.
Does intravenous vaccination with self-killing BCG lead to development of an immune response that protects against Mycobacterium tuberculosis?
Authors: Smith Alexander A , Rodgers Mark A , Bigbee Carolyn M , Schnappinger Dirk , Ehrt Sabine , Flynn JoAnne L
Published: August 2023

The use of intradermal BCG offers limited protection against tuberculosis. Intravenous (IV) administration of BCG has shown to produce robust protection against infection in a non-human primate model of tuberculosis. However, the duration of exposure and time required to elicit an efficacious, protective response is unknown and safety of IV administration of live BCG remains a concern. Here we investigated whether a self-limiting strain of BCG (killswitchBCG; ksBCG) could induce protective responses in macaques. ksBCG uses two transcriptionally repressible lysins; the inducer doxycycline is required for repression, with the absence of inducer leading to BCG death. Our first study demonstrated the robust in vivo killing of ksBCG once doxycycline is stopped IV ksBCG elicited a robust, multicellular immune response in airways and lung up to 8 weeks, similar to standard BCG. In the current study we are assessing protection conferred against Mycobacterium tuberculosis with IV ksBCG. Mauritian cynomolgus macaques (MCM) were vaccinated intravenously with ksBCG. 5 months post vaccination, animals were challenged with M. tuberculosis and later euthanized 12 weeks post infection. Bronchoalveolar lavages were routinely taken throughout the study, and alongside tissue samples, analyzed using spectral flow cytometry. The presence of multiple cell types (CD4, CD8, and gd T cells, B cells, NK cells), cytokines (IFNg, IL-2, IL-17, TNF) and cytotoxic molecules (granzymes B and K, perforin, granulysin), as well as M. tuberculosis bacterial burden will be assessed to determine the level of protection provided by IV ksBCG.

NIH R01 AI143788
Repetitive vaccination with intravenous BCG is safe and immunogenic in naïve and SIV-infected macaques
Authors: Lehman Chelsea C , King Hannah A , Pokkali Supriya , Bucsan Allison N , Seder Robert , Roederer Mario , Bolton Diane , Darrah Patricia A
Published: August 2023

Tuberculosis (TB) is the leading cause of death in people with HIV (PWH) on or off antiretroviral therapy (ART); thus, new TB vaccine candidates should be evaluated in the setting of HIV. Mycobacterium bovis Bacillus Calmette-Guerin (BCG), the live attenuated bacterial TB vaccine given intradermally (ID) at birth, is contraindicated in PWH. We recently demonstrated that intravenous (IV) delivery of BCG elicits superior immunity and protection compared to ID delivery against Mycobacterium tuberculosis (Mtb) challenge in rhesus macaques. To assess IV BCG in an animal model of HIV infection, we immunized SIVmac251-infected macaques on suppresive ART (6m) and uninfected controls. Rhesus macaques received three doses of IV BCG, 8wks apart, and were monitored for the presence of BCG colony forming units (CFU) in peripheral blood and bronchoalveolar lavage (BAL) and Mtb-specific T cell responses in BAL. Repetitive IV BCG immunization was well-tolerated in SIV-infected and uninfected animals, with no difference in clinical parameters or BCG CFU in blood or BAL. T cell responses after IV BCG were similar, with both groups displaying a 10–50 fold increase in BAL T cell abundance after initial dose. Mtb-specific CD4 and CD8 responses peaked after the initial IV BCG dose and were maintained by subsequent immunizations for at least 6m. These data indicate that IV BCG is safe and immunogenic in SIV-infected, ART-suppressed macaques and that IV BCG boosting maintains T cell responses at the site of infection.

NIH Intramural Program
Characterizing the Plasma IgG Antibody Repertoire of Malaria-Naïve United Kingdom Adults Vaccinated with Novel Blood Stage Vaccine RH5.1/AS01B
Authors: Marchioni Jeffrey , Seeger Allison , Barrett Jordan R , McHugh Kirsty , Kain Jessica , MacGill Randall , Georgiou George , Draper Simon J , Lavinder Jason J , Ippolito Gregory C
Published: August 2023

Plasmodium falciparum, the most lethal species of human malaria, is susceptible to vaccine-induced antibodies, yet current efforts are challenged by limited efficacy, lack of durability, and parasite evasion. A promising blood-stage vaccine antigen, Reticulocyte Binding Protein Homologue 5 (RH5), has low polymorphism frequencies and uses a non-redundant red blood cell (RBC) invasion pathway. The Draper Lab (Oxford University) clinically tested RH5.1, an engineered variant of RH5, in AS01B adjuvant (GSK) designed to boost long-lasting, protective antibody titers.

RH5-specific B cell receptors (BCRs) from United Kingdom malaria–naïve adult volunteers vaccinated with RH5.1/AS01B were sequenced. Monoclonal antibodies (mAbs) were found to inhibit in vitro growth of RBC-invading parasite merozoites. Notably, the polyclonal plasma IgG of the volunteers exhibited high neutralizing potency, exceeding that of individual mAbs from the same donors, which raises the question: what is the disconnect between the BCR and circulating IgG repertoires? To examine this further, our lab completed BCR sequencing coupled with plasma IgG proteomics to characterize the plasma antibody repertoires of volunteers.

Plasma mAbs were identified that bind to known growth inhibitory epitopes, including those that block binding between RH5 and its RBC coreceptor, CD147, alongside a population of non-neutralizing, synergizing mAbs. Additionally, non-neutralizing mAbs targeting cryptic epitopes on the N- and C-terminus were found at consistently high levels. This dynamic between neutralizing and non-neutralizing plasma mAbs suggests a role for non-neutralizing mAbs, which should be further explored in future RH5 vaccine engineering.

Supported by grants from U.S. Agency for International Development (USAID) contract no. 7200AA20C00017 and the National Science Foundation Graduate Research Fellowship Program (NSF GRFP).
Evaluation of the time needed for BCG to be alive to protect against tuberculosis in intravenously BCG vaccinated SIV+ macaques
Authors: Jauro Solomon , Larson Erica C , Maiello Pauline , Rodgers Mark A , Ameel Cassaundra L , Gleim Janelle L , Sakal Jennifer , Kracinovsky Kara , Lin Philana Ling , Flynn JoAnne L , Scanga Charles A
Published: August 2023

Effective means of improving Bacille Calmette-Guérin (BCG) immunogenicity to protect against pulmonary tuberculosis (TB) is needed, especially among people living with HIV. Intravenous (IV) administration of BCG previously showed significant enhancement of immune responses and conferred ~75% protection against TB. Using our SIV/M. tuberculosis (Mtb) coinfection model (to mimic HIV/Mtb coinfection) in Mauritius cynomolgus macaques (MCM), we evaluated the length of time necessary for live BCG to elicit a protective immune responses by varying the timing of anti-BCG drug (isoniazid, rifampicin, and ethambutol: HRE) initiation. MCM were infected with SIVmac239 and randomly assigned into 4 different vaccination groups 16 weeks after SIV infection: intradermal BCG (BCG ID No HRE), BCG IV with HRE started 1 week post BCG (BCG IV 1wk HRE), BCG IV with HRE started 3 weeks post BCG (BCG IV 3wks HRE), and BCG IV without HRE treatment (BCG IV No HRE). The HRE treatment lasted for 8 weeks. Airways samples were collected by bronchoalveolar lavage (BAL), whereas lung tissue and lymph node were harvested at necropsy in a subset of BCG ID and BCG IV 3wks vaccinated MCM 20 weeks after BCG but before (Mtb) challenge. The remaining MCM were challenged with Mtb Erdman strain (~15 CFU). CD4 and CD8 T cell levels increased in BAL in all IV vaccinated MCM. CD4 T cells producing IFNg, IL-2, IL-17, and TNF peaked after BCG and were maintained until necropsy. There were increased tissue-resident T cells and cytokine production in lungs of IV vaccinated MCM compared to BCG ID. No signs of BCG dissemination were apparent in any IV vaccinated MCM. We are currently evaluating protection against infection and disease in the SIV+ MCM in each vaccination group.

Supported by grant NIH (R01 AI155345)
Antigen experience history directs distinct functional states of CD8+ CAR T cells during the anti-leukemia response
Authors: DeGolier Kole R , D’Antonio Marc , Danis Etienne , Yarnell Michael , Kohler Mark Eric , Kedl Ross , Scott-Browne James , Fry Terry
Published: August 2023

Chimeric antigen receptor (CAR) T cells are highly effective in treating hematologic cancers, but patients can relapse due to downregulation of the CAR-targeted antigen, leading to the hypothesis that CAR T cells require high density of target antigen to be effective. Non-engineered CD8+ memory T cells generate more rapid and robust effector T cell responses upon secondary infection, relative to their naïve counterparts, and can display higher antigen sensitivity. Our objective was to characterize the role of prior CD8+ T cell antigen experience (AgEx) in dictating functional CAR-mediated anti-tumor responses. To study the effect of T cell AgEx history on CD8+ CAR T cell (CAR8) function in vivo, we used a vaccine model to control prior T cell AgEx history, and a syngeneic adoptive transfer model with murine anti-CD19 CAR8 targeting murine leukemia engineered to express a low or high level of CD19. We analyzed CAR8 expansion and leukemia burden using flow cytometry and performed ATACseq and RNAseq at timepoints pre- and post-CAR transduction and after reinfusion of CAR8 into leukemia-bearing mice to ask whether CAR8 function was determined by the epigenetic and/or transcriptomic state of the starting cell population. We found that starting cell state dictated in vivo CAR8 effector functions, expansion, and differential chromatin accessibility in regions containing binding motifs for AP-1, Runx, Tcf and other transcription factor (TF) families. Motif chromatin accessibility and gene expression of many TFs showed distinct patterns temporally. We conclude that CAR8 maintain functional traits epigenetically imprinted in the starting cell and predict these analyses could inform methods to transcriptionally modulate CAR T cell function.
Sensitizing solid tumors to respond to Cytokine-induced apoptosis and CAR T-cell therapy
Authors: Guttormson Matthew C , Ting Adrian
Published: August 2023

Chimeric antigen receptor (CAR) T-cells are T-cells engineered with synthetic receptors that directs their specificity and function toward an antigen of choice. This application has been highly successful in treating a variety of blood-borne cancers such as B-cell lymphomas. CAR T-cells against solid tumors have been less efficacious, potentially due to the resistance of tumor cells to T cell-mediated cytotoxicity. Current paradigm suggests that tumor cells are killed by perforin/granzyme-secreting CD8 cytotoxic T cells (CTLs). However, activated CD8 T cells also produce TNF and IFNg, two cytokines that have been reported to have anti-tumor function, but it is not clear whether these cytokines can directly kill tumor targets. It has been reported that defects in kinases in the TNFR1 signaling pathway can sensitize cells to TNF-induced death. Therefore, we hypothesize that by inactivating these kinases, we can sensitize solid tumors to killing by CD8 CAR T-cells producing TNF and IFNg. We intend to conduct proof-of-concept studies to show that sensitizing tumor cells to cytokine-mediated cytotoxicity will enhance the effectiveness of CAR T-cell therapy against solid tumors.

Institutional Funding
Predicting the therapeutic efficacy of TLR stimulated macrophages for cancer treatment
Authors: Kurt Robert A , Newman Thalia , Liew Chun Wai
Published: August 2023

Macrophages play a variety of roles in immunity including both pro- and anti-inflammatory functions, which can be controlled by toll-like receptor (TLR) stimulation. Previously, we generated a computational model of interconnected TLR signaling cascades and validated the model using experimental data from the THP-1 monocyte cell line. This project aimed to adapt the computational model to provide insights into the activation of bone marrow-derived macrophages (BMDM) to enable the accurate prediction of BMDM therapeutic efficacy following TLR stimulation. Initially, we quantitated expression of genes encoding TLR signaling proteins in BMDM and THP-1 cells and found distinct differences between the cell types. For instance, IRAK2 was expressed in BMDM at a level over 150 times the expression level in THP-1 cells, while other genes, such as TLR4, were expressed at relatively similar levels. Next, these data were used to adapt the computational model to BMDM. In order to determine whether the model predicted the optimal way to activate the BMDM, the cells were treated with lipopolysaccharide and flagellin and IL-1b production was quantified by ELISA. The data revealed that the model did not predict the optimal way to achieve pro-inflammatory cytokine expression in the BMDM. We are currently altering the model to improve its ability to predict pro-inflammatory cytokine expression following TLR stimulation. In addition, studies are underway exploring the therapeutic efficacy of the TLR-stimulated BMDM using a murine mammary carcinoma model. The ultimate goal of this project is to produce a computational model capable of predicting how BMDM respond to TLR stimulation and to use the model to predict the anti-tumor efficacy of the BMDM.
Triple checkpoint blockade, but not anti-PD1 alone, enhances the efficacy of engineered adoptive T cell therapy in advanced ovarian cancer
Authors: Anderson Kristin G , Su Yapeng , Burnett Madison , Bates Breanna , Suarez Magdalia Rodgers , Voillet Valentin , Gottardo Raphael , Greenberg Philip D
Published: August 2023

Less than 50% of ovarian cancer patients survive five years after diagnosis. This rate has changed little in the last 30 years, highlighting the need for novel therapies. A promising strategy employs T cells engineered to target tumor-expressed proteins. Mesothelin (Msln) is overexpressed in ovarian cancers, contributes to the malignant and invasive phenotype and has limited expression in healthy cells, making it a candidate immunotherapy target.

We used the immune-competent ID8VEGF mouse model of advanced ovarian cancer to show that T cells engineered to express a Msln-specific T cell receptor (TCRMsln) can preferentially accumulate within established tumors, delay tumor growth and significantly prolong mouse survival. However, elements in the tumor microenvironment limited engineered T cell function. Tumor-bearing mice were therefore treated with TCRMsln T cells plus PD1, Tim-3 and/or Lag-3 checkpoint-blocking antibodies, alone or in combination. Triple checkpoint blockade, but not single- or double-agent treatments, dramatically increased effector cytokine production by intratumoral TCRMsln T cells. Single cell RNA-sequencing revealed gene expression changes in intratumoral TCRMsln T cells and myeloid cells consistent with immune activation and inflammation. Moreover, combining adoptive immunotherapy with triple checkpoint blockade prolonged survival in treated mice, relative to TCRMsln with or without single- or double-agent checkpoint blockade.

These results suggest that disrupting multiple immune-inhibitory pathways simultaneously, which can be more safely pursued in a cell intrinsic form through genetic engineering, may be necessary to improve outcomes for ovarian cancer patients.
XCL1 facilitates the interaction with CD8+ T-cells and XCR1+ DCs within the Cervical Lymph Nodes during WNV encephalitis
Authors: De La Torre Kristen , Durrant Douglas M
Published: August 2023

West Nile Virus (WNV), a neurotropic flavivirus, causes encephalitis, especially in elderly and immunocompromised individuals. Previous studies have demonstrated that CNS-localized dendritic cells (DCs) protect against WNV neuroinvasive disease by establishing antiviral T-cell responses in the CNS. In this study, we investigated the role of the XCL1-XCR1 interaction since its primary function is to facilitate DC-mediated CD8+ T cell immunity. XCL1, also known as lymphotactin, is expressed by various immune cells, including activated CD8+ T cells, and binds to XCR1, a chemokine receptor expressed predominately by a subset of DCs. Following subcutaneous infection with WNV, XCL1 expression increased within the CNS on day 6 post-infection after local viral replication began. In contrast, the expression of its receptor, XCR1, increased by day 9 post-infection in wild-type (WT) mice. We found that WNV-infected XCR1-deficient (XCR1−/−) mice demonstrated increased symptomatic disease and mortality in conjunction with the increased viral burden correlating with decreased CD8+ T-cell recruitment and activation at day 12 post-infection in the CNS compared with their WT counterparts. We found that XCR1+ DCs were present in the cervical lymph nodes at days 6 and 9 post-infection and in the CNS at day 12 post-infection. Taken together, these results suggest that the XCL1-XCR1 interaction promotes viral clearance and protection against WNV neuroinvasive disease.

NIH-SC3GM130472
Studies in humans and mice reveal a critical role for CCR2 in trafficking of pDCs during infection with SARS-CoV-2
Authors: Zhang Howard H , Lu Xinping , Garner Davis , Parween Farhat , Singh Satya , Majumdar Shamik , Weaver Joseph , Stein Sydney , Lesho Patricia , Damcott Coleen , Lutfi Forat , Petrick Kendra , Rock Peter , Chertow Daniel , Kelsall Brian L , Murphy Philip M , Dahiya Saurabh , Hardy Nancy , Farber Joshua
Published: August 2023

Although GWAS have implicated chemokine receptors in affecting disease severity, the role of chemokines in COVID-19 pathogenesis has not been defined. To bridge this gap, we surveyed expression of 14 chemokine receptors on blood leukocytes from 19 hospitalized COVID-19 patients vs. healthy controls. Among many differences between patients and controls, we found that CCR2, which we have shown mediates lymphocyte transendothelial migration, was down on patient MAIT cells and pDCs, whose numbers in blood are depressed in COVID-19. If a chemokine receptor mediates extravasation, lower levels of the receptor on blood cells could result from efficient, and thereby preferential migration of leukocytes expressing higher levels of the receptor into infected tissue. In support of this mechanism, we found enrichment of CCR2high cells among MAIT cells migrating to the CCR2 ligand, CCL2, in vitro. To test if CCR2 mediates leukocyte extravasation in COVID-19 we infected transgenic K18-hACE2 Ccr2 reporter mice that were Ccr2+/RFP (Ccr2 sufficient) or Ccr2RFP/RFP (Ccr2 deficient) with SARS-CoV-2 intranasally and analyzed pDCs. Blood pDCs were RFP+ and their numbers were decreased in Ccr2RFP/RFP mice at baseline. Over seven days after infection with SARS-CoV-2 (LD50), pDCs increased and then decreased in the blood, while increasingly progressively in lungs in both Ccr2+/RFP and Ccr2RFP/RFP mice. However, absolute numbers of lung pDCs and ratios of lung/blood pDCs were markedly diminished, as was survival, in Ccr2RFP/RFP mice. We propose that increased mortality in Ccr2-deficient mice may be due in part to inadequate numbers of lung pDCs, leading to local deficiencies in type 1 and III interferons, which are critical for defense against SARS-CoV-2.
Diabetic wound keratinocytes induce macrophage JMJD3-mediated Nlrp3 expression via IL1R signaling
Authors: Wolf Sonya , Audu Christopher , Moon Jadie Y , Joshi Amrita D , Melvin William J , Barrett Emily C , Mangum Kevin D , Gallagher Katherine A
Published: August 2023

Type 2 diabetic (T2D) wound Macrophages (Mϕs) display a delayed chronic inflammatory state that prevents the resolution of wound inflammation. Increased NLRP3 inflammasome gene expression and activation in Mϕs contributes to impaired wound healing in T2D and is induced by diabetic wound environment factors, yet the molecular mechanisms regulating this are unclear. Our lab has shown that keratinocytes regulate Mϕ phenotype during wound healing by producing proinflammatory cytokines. Thus, this project explores the molecular mechanisms by which keratinocytes drive increased Nlrp3 expression in diabetic wound Mϕs. Here, we found that Nlrp3 expression is increased in diabetic wound Mϕs late post-injury, and this can be induced by stimulation of Mϕs with diabetic wound keratinocyte supernatant. Further, we found that stimulation of Mϕs with diabetic wound keratinocyte supernatant decreased H3K27me3 deposition at the Nlrp3 promoter. Using genetically engineered mice (Jmjd3 fl/fl lyz2cre), we demonstrate that JMJD3, a demethylase specific for H3K27, controls Mϕ-mediated Nlrp3 expression and inflammasome activation during wound healing. Importantly, we show that diabetic wound keratinocytes induce Jmjd3 expression in Mϕs via IL1R/MyD88 upstream signaling. Interestingly, we found that IL1α is increased in human and murine wound diabetic keratinocytes compared to non-diabetic controls and stimulation of Mϕs with IL1α increases Nlrp3 expression via a JMJD3-mediated mechanism. Thus, our data suggest a role for keratinocyte IL1α in driving increased Nlrp3 expression in diabetic wound Mϕs via JMJD3. Continued investigation into the keratinocyte-Mϕ axis is key to developing novel treatments for non-healing T2D wounds.

P30 DK089503, 1K99DK133828-01, 5K12 GM111725