Citrullination of LL-37 as a mechanism that selectively controls immunostimulatory potential of DNA

LL-37, jedyna ludzka katelicydyna, jest amfipatycznym, kationowym peptydem bakteriobójczym, który zawiera 37 reszt aminokwasowych. Dojrzały peptyd uwalniany jest z 18 kDa białka prekursorowego (katelicydyny) przez proteazę-3 w neutrofilach lub przez proteazy serynowe z rodziny kallikrein w keratynocytach. Oprócz zdolności bezpośredniego zabijania mikroorganizmów, LL-37 pełni funkcję immunomodulacyjną. Jedną z nich jest tworzenie kompleksów z ujemnie naładowanymi oligonukleotydami pochodzącymi zarówno z obumierających komórek gospodarza, jak i patogenów. Interakcja ta ułatwia rozpoznawanie DNA przez receptory wewnątrzkomórkowe, takie jak TLR-9 prowadząc do indukcji odpowiedzi immunologicznej. W ostatnim czasie udokumentowano, że zdolność peptydu LL-37 do pełnienia funkcji bakteriobójczych i immunomodulacyjnych zależy w dużym stopniu od jego modyfikacji potranslacyjnych, wśród których deiminacja odgrywa wyjątkową rolę. Struktura pierwszorzędowa LL-37 zawiera pięć reszt argininy (Arg), które są wydajnie cytrulinowane przez deiminazy peptydylo-argininowe (PADs), enzymy, które są zależnymi od wapnia hydrolazami katalizującymi konwersję dodatnio naładowanej argininy do neutralnej cytruliny. W związku z powyższym w niniejszej pracy skupiono się na roli deiminacji peptydu LL-37 w regulacji odpowiedzi zapalnej na kwasy nukleinowe. Uzyskane wyniki dowiodły, że cytrulinacja LL-37 katalizowana przez PAD2 ogranicza zależne od peptydu pochłanianie DNA przez fagocyty. Co ciekawe, karbamylacja peptydu (homocytrulinacja reszt lizyny) nie ma wpływu na jego interakcję z DNA, tak długo jak karbamylowany peptyd nie zostanie poddany działaniu PAD2. W badaniach zastosowano również syntetyczny peptyd LL-37 z resztami Arg zastąpionymi homoargininą (hArg-LL-37), która jest niewrażliwa na proces enzymatycznej deiminacji przez PADy. Dane te wykazały, że wiązanie peptydu LL-37 z DNA, a tym samym jego działanie immunoregulacyjne względem fagocytów nie zależy wyłącznie od ładunku dodatniego peptydu, ale od prawidłowego rozmieszczenia guanidynowych łańcuchów bocznych argininy w natywnej sekwencji peptydu. Aby zbadać, czy powyższe zjawisko odgrywa rolę w stanie zapalnym przeprowadzono badania z wykorzystaniem zewnątrzkomórkowych pułapek neutrofilowych (NETs, ang. neutrophil extracellular traps). Tworzenie tych struktur, zbudowanych z nici genomowego DNA udekorowanych białkami pochodzącymi z ziaren azurofilnych, w tym LL-37, zależy od aktywności PAD4. Szczegółowa analiza składu NETs i ich wpływu na profesjonalne fagocyty sugeruje, że katelicydyna/LL-37 jest cytrulinowana przez PAD4 podczas tworzenia pułapek neutrofilowych. Proces ten odgrywa istotną rolę w kontroli potencjału zapalnego NETs, zwłaszcza w regulacji odpowiedzi immunologicznej na uwolnione z obumierających komórek DNA, które jest rozpoznawane przez receptor TLR9. Przebieg procesu cytrulinacji w warunkach in vivo potwierdzają wyniki analizy serologicznej surowic pochodzących od pacjentów cierpiących na przewlekłe choroby zapalne, takie jak: paradontozę i reumatoidalne zapalenie stawów, w których wykazano podwyższony poziom przeciwciał rozpoznających cytrulinowaną formę peptydu LL-37. Ostatecznie, dowiedziono, że cytrulinowana forma peptydu LL-37 zwiększa powstawanie NETs, co sugeruje mechanizm pozytywnego sprzężenia zwrotnego. Podsumowując, cytrulinacja peptydu LL-37 odgrywa kluczową rolę w jego immunomodulacyjnym działaniu przeciwdziałając utracie tolerancji gospodarza na własne cząsteczki, w tym DNA.LL-37, the only human cathelicidin, is an amphipathic, cationic antimicrobial peptide that contains 37 amino acids residues. The mature peptide is released from an 18-kDa precursor protein (cathelicidin) by protease-3 in neutrophils or by serine proteases of the kallikrein family in keratinocytes. Besides playing antimicrobial role, LL-37 exerts several immunomodulatory functions that regulate host responses to pathogens. Among them is formation of complexes with negatively charged oligonucleotides derived from both dying host cells and pathogens. Such interaction facilitates the recognition of DNA by intracellular receptors such as TLR9, thereby stimulating the immune response. It was recently documented that the ability of peptide to execute its bactericidal and immunomodulatory functions strongly depends on posttranslational modifications, among which enzymatic deimination (citrullination) of Arg residues plays a unique role. The primary structure of LL-37 contains five arginine (Arg) residues that are effectively citrullinated by peptidyl-arginine deiminases (PADs), enzymes that are calcium-dependent hydrolases catalyzing the conversion of positively charged arginine to neutral citrulline. Therefore, presented study focused on the role of LL-37 deimination in the regulation of the host immune response to nucleic acids. Presented data revealed that citrullination of LL-37 by PADs hindered peptide-dependent DNA uptake and sensing by pDCs. In contrast, carbamylation of the peptide (homocitrullination of lysine residues) had no effect on its interaction with DNA. This activity was abolished by citrullination of Arg residues in the peptide by PAD2. We implemented in the study the synthetic peptide LL-37 with Arg residues substituted by homoarginine (hArg-LL-37), which is insensitive to enzymatic deimination. Using such peptide, we observed that regardless of PAD2 treatment hArg-LL-37 preserved full biological activity. These data have showed that peptide binding to DNA and its immunoregulatory effects on myeloid cells do not depend entirely on the positive charge of LL-37 but on the proper distribution of guanidium side chains of Arg (or homo-Arg) in the native peptide sequence. To explore if above phenomenon plays a role in inflammatory conditions, we introduced in the study neutrophil extracellular traps (NETs). Formation of those structures, composed of genomic DNA and decorated with azurophilic proteins including LL-37, depends on PAD4 activity. Detailed analysis of NETs composition and their influence on myeloid cells suggest that cathelicidin/LL-37 is citrullinated by PADs during NETs formation. Such process makes a marked contribution to the inflammatory potential of NET structures especially in regulating the immunostimulatory response of cell-free DNA, which is mediated by TLR9. Such conclusion was additionally supported by an elevated antibody response against citrullinated LL-37 in blood serum obtained from patients suffering from chronic inflammatory diseases (periodontitis, rheumatoid arthritis). Finally, obtained data revealed that citrullinated LL-37 itself enhances the generation of NETs formation suggesting the mechanism of positive feedback. Taken together, by preventing the breakdown of tolerance to self-molecules, citrullination of LL-37 plays a critical role in immunomodulation of the host response

The development of a health-related quality of life assessment tool for adolescent and young adult survivors of central nervous system tumors

Thesis (M.A.)--Boston UniversityCentral nervous system (CNS) tumors are the most common form of solid tumor in children and young adults. Medical advancements have increased survival rates dramatically for people diagnosed with CNS tumors, but long-term disease-and treatment-related sequelae remain significant issues for survivors. The chronic health conditions and vulnerability to late effects that this population of survivors experiences are different from that of survivors of other forms of cancer due to the nature and location of CNS tumors. Health-related quality oflife (HRQoL) measurement has become a useful and significant tool for tracking the progress and well-being of patients long-term. This paper examines HRQoL specifically for adolescents and young adults (AYA) who are CNS tumor survivors, and describes the findings of a research study conducted to develop the Health Related Quality of Life Evaluation in Survivorship of CNS Tumors (QUEST CNS) instrument. METHODS: Five focus groups were conducted with 19 AYA survivors of CNS tumors in November and December 2010. Transcripts were coded and analyzed to identify the major domains of HRQoL to be included in the QUEST CNS instrument being developed. Separately, a review of existing HRQoL instruments was conducted to identify major domains in the literature and these instruments. 324 potential items were generated from the focus group findings and instrument review, and the list was electronically rated by a group of 8 experts in the fields of psychology, oncology, and hematology. Content validity ratios were determined for each item, and the list of items was amended accordingly. 165 items were removed, leaving 159 items for the preliminary QUEST CNS instrument. [TRUNCATED

A high-throughput screen identifies that CDK7 activates glucose consumption in lung cancer cells.

Elevated glucose consumption is fundamental to cancer, but selectively targeting this pathway is challenging. We develop a high-throughput assay for measuring glucose consumption and use it to screen non-small-cell lung cancer cell lines against bioactive small molecules. We identify Milciclib that blocks glucose consumption in H460 and H1975, but not in HCC827 or A549 cells, by decreasing SLC2A1 (GLUT1) mRNA and protein levels and by inhibiting glucose transport. Milciclib blocks glucose consumption by targeting cyclin-dependent kinase 7 (CDK7) similar to other CDK7 inhibitors including THZ1 and LDC4297. Enhanced PIK3CA signaling leads to CDK7 phosphorylation, which promotes RNA Polymerase II phosphorylation and transcription. Milciclib, THZ1, and LDC4297 lead to a reduction in RNA Polymerase II phosphorylation on the SLC2A1 promoter. These data indicate that our high-throughput assay can identify compounds that regulate glucose consumption and that CDK7 is a key regulator of glucose consumption in cells with an activated PI3K pathway

Testosterone replacement and improved memory

Dr Lyon is Associate Professor of Family Medicine, Dr Wong is an instructor and fellow in family medicine, and Ms DeSanto is a clinical librarian, all at the University of Colorado in Denver.Clinical Inquires question: Does testosterone replacement improve cognition in memory-impaired older men with low testosterone? Evidence-based answer: Testosterone therapy does not appear to improve cognition in older men with low testosterone and memory impairment (strength of recommendation B, based on systematic reviews of small, low-quality randomized controlled trials [RCTs] and individual, small RCTs).Corey Lyon, DO; Alicia Wong, MD, MPH, MA; Kristen DeSanto, MSLS, MS, RD, AHIPCan Fam Physician 2021;67:e9. DOI: 10.46747/cfp.6701e9Includes bibliographical reference

Generating health technology assessment evidence for rare diseases

Objectives: Rare diseases are often heterogeneous in their progression and response to treatment, with only a small population for study. This provides challenges for evidence generation to support HTA, so novel research methods are required. Methods: Discussion with an expert panel was augmented with references and case studies to explore robust approaches for HTA evidence generation for rare disease treatments. Results: Traditional RCTs can be modified using sequential, three-stage or adaptive designs to gain more power from a small patient population or to focus trial design. However, such designs need to maintain important design aspects such as randomization and blinding and be analyzed to take account of the multiple analyses performed. N-of-1 trials use within-patient randomization to test repeat periods of treatment and control until a response is clear. Such trials could be particularly valuable for rare diseases and when prospectively planned across several patients and analyzed using Bayesian techniques, a population effect can be estimated that might be of value to HTA. When the optimal outcome is unclear in a rare disease, disease specific patient reported outcomes can elucidate impacts on patients’ functioning and wellbeing. Likewise, qualitative research can be used to elicit patients’ perspectives, with just a small number of patients. Conclusions: International consensus is needed on ways to improve evidence collection and assessment of technologies for rare diseases, which recognize the value of novel study designs and analyses in a setting where the outcomes and effects of importance are yet to be agreed.</p

CONTROL OF THE CA2+ - NFAT-IL-2 PATHWAY BY LRRK2 IN ASPERGILLUS-STIMULATED DENDRITIC CELLS

Ph.DDOCTOR OF PHILOSOPH

Experience of BRCA1/2 mutation-negative young women from families with hereditary breast and ovarian cancer: a qualitative study

BACKGROUND: Little is known about the experience of young women who become aware of their parent’s BRCA1 or BRCA2 (BRCA) mutation status as adolescents or young adults. There is also currently a gap in the literature pertaining to those who are found to be negative for their familial mutation. We aimed to investigate the experience of these mutation-negative young women from hereditary breast and ovarian cancer (HBOC) families. METHODS: Using a semi-structured questionnaire we interviewed 8 women. All of the women were non-carriers of their familial mutation and had learned of the mutation in their family as adolescents or young adults at least 6 months prior to undergoing genetic testing. All interviews were audio recorded, transcribed, and independently analyzed by the investigators. This was followed by an in-depth cross-case analysis, enabling the formulation of emergent themes. RESULTS: The women’s age ranged from 22 to 37 years old and all were of Ashkenazi Jewish descent. Prominent emergent themes from the interviews included the impact of how and when the familial mutation status was disclosed, the factors influencing when a young woman chooses to undergo predictive genetic testing, the predictors of post-test adjustment and risk perception, as well as the impact of familial cancer experience versus the familial mutation. CONCLUSIONS: By eliciting detailed patient narratives we have begun to show that this generation of BRCA mutation-negative young women is likely still affected by the degree of cancer history in their family, even with their understanding of the genetic contribution to disease. Larger studies with tightened participant characteristics, as well as studies involving women from different cultural backgrounds, are needed to further define the experience and needs of true negative young women from HBOC families

Interleukin-2 Production by Dendritic Cells and its Immuno-Regulatory Functions

Dendritic cells (DCs) are uniquely potent antigen presenting cells that acquire microbial products and prime adaptive immune responses against pathogens. Furthermore, DCs also play a key role in induction and maintenance of tolerance. Although numerous studies have assessed the diverse functions of DCs, many unanswered questions remain regarding the molecular mechanisms that DCs use to achieve immunoregulation. While not widely regarded as a significant provider of T-cell growth factors, DCs have previously been identified as a potential source of IL-2 cytokine. Recent research indicates that microbes are the most effective stimuli to trigger IL-2 production in DCs by activating the calcineurin/NFAT signaling pathway. Herein we describe recent insights into the production and function of IL-2 cytokine and IL-2 receptor in DCs early after stimulation through pattern recognition receptors. These findings clarify how DCs fine-tune effector and regulatory responses by modulating IL-2 production in both tolerance and immunity

Alteración en los Hábitos del Consumidor Saltillense a Consecuencia del COVID-19

El estudio pretende indagar sobre la modificación en el comportamiento del consumidor saltillense debido a la contingencia por la epidemia del nuevo coronavirus COVID-19. El trabajo incluyó a 354 participantes adultos residentes en la Ciudad de Saltillo, Coahuila, México. La muestra está conformada por 195 mujeres, es decir, un 55.1% y 159 hombres lo que constituye un 44.9%. Se realiza un trabajo de tipo Cuantitativo, exploratorio, descriptivo y transversal no experimental. Las preguntas son categóricas y la escala de respuesta es tipo Likert del 1 al 7, validando un instrumento de 25 elementos obteniendo un Alfa de Cronbach .786. Con base en las variables analizadas se infiere que los saltillenses se han vuelto consumidores más racionales, prefiriendo alimentos caseros, cuidando sus gastos, preocupándose por el futuro y extrañando la experiencia de comprar en tiendas físicas favoreciendo las ventas por internet y el servicio a domicilio. Y concluye que existe un nuevo perfil de consumidor no solo en Saltillo, sino a nivel mundial, que procura el bienestar en su salud, modificando hábitos alimenticios y conductuales adaptándose a esta nueva normalidad. &nbsp; The study aims to investigate the modification in the behavior of the Saltillo consumer due to the contingency caused by the epidemic of the new coronavirus COVID-19. The work included 354 adult participants residing in the City of Saltillo, Coahuila, Mexico. The sample is made up of 195 women, that is, 55.1% and 159 men, which constitutes 44.9%. A quantitative, exploratory, descriptive and non-experimental cross-sectional work is carried out. The questions are categorical and the response scale is Likert type from 1 to 7, validating an instrument of 25 elements obtaining a Cronbach's Alpha .786. Based on the variables analyzed, it can be inferred that the people of Saltillo have become more rational consumers, preferring homemade foods, taking care of their expenses, worrying about the future and missing the experience of buying in physical stores, favoring internet sales and home delivery. And it concludes that there is a new consumer profile not only in Saltillo, but also worldwide, which seeks the well-being in their health, modifying eating and behavioral habits, adapting to this new normal

Loss of Gata6 causes dilation of the hair follicle canal and sebaceous duct

The uppermost aspect of the hair follicle, known as the infundibulum or hair canal, provides a passageway for hair shaft egress and sebum secretion. Recent studies have indicated that the infundibulum and sebaceous ducts are lined by molecularly distinct differentiated cells expressing markers including Keratin 79 and Gata6. Here, we ablated Gata6 from the skin and observed dilation of both the hair canal and sebaceous ducts, independent of gender and hair cycle stage. Constitutive loss of Gata6 yielded only a mild delay in depilation‐induced entry into anagen, while unperturbed mutant mice possessed overtly normal skin and hair. Furthermore, we noted that Keratin 79 and Gata6 expression and localization did not depend upon each other. Our findings implicate Gata6 in maintaining the upper hair follicle and suggest that regulation of this transcription factor may be compromised in pathologies such as acne or infundibular cystic diseases that are characterized by abnormal expansion of this follicular domain.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/149235/1/exd13757_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/149235/2/exd13757-sup-0001-FigS1-S9.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/149235/3/exd13757.pd