Effects of Photobiomodulation Associated with Platelet-Rich Plasma in Acute Rheumatoid Arthritis Induced in Female Wistar Rats' Knee.

Objective: Rheumatoid arthritis causes inflammation, pain, and joint degradation, necessitating treatment with anti-inflammatory drugs and corticosteroids, posing various challenges. We aimed to evaluate the effects of photobiomodulation (PBM) at two different doses associated to platelet-rich plasma (PRP) in an in vivo model of induced acute arthritis in Wistar rats' knee. Methods: Eighty-four Wistar rats were assigned into seven groups, including animals treated with PBM and/or PRP. On day 0, arthritis was induced in sham and treated groups through the intra-articular injection of zymosan (200 µg). Twenty-four hours after induction, the PBM groups were treated with an AsGaAl laser, whereas the PRP-treated groups received intra-articular injections with a concentration of 8 × 105 platelets obtained from another four animals. After 3 days, the animals were euthanized, and the interleukin (IL)-6 and complement C3 gene and protein expression levels were analyzed. Statistical analysis was performed using the mean ± SD with analysis of variance and Tukey's posttest, with a significance level set at 5% (p < 0.05). Results: Synovial inflammation decreased in PBM-treated groups; however, PRP alone showed no significant difference. Gene expression analysis revealed a significant difference in IL-6 and C3 levels in the PBM and PBM+PRP-treated groups. Meanwhile, the PRP alone group exhibited significance for IL-6. Moreover, the PBM and PBM+PRP-treated groups showed a significant difference in C3 protein expression levels, whereas the PRP alone group showed no difference. Conclusion: The increase in cellular activity in the synovial membrane and the decrease protein expression levels are owing to the reduction in proinflammatory mediators following PBM therapy.

Oxylipins and metabolites from pyroptotic cells act as promoters of tissue repair.

Pyroptosis is a lytic cell death mode that helps limit the spread of infections and is also linked to pathology in sterile inflammatory diseases and autoimmune diseases1-4. During pyroptosis, inflammasome activation and the engagement of caspase-1 lead to cell death, along with the maturation and secretion of the inflammatory cytokine interleukin-1ß (IL-1ß). The dominant effect of IL-1ß in promoting tissue inflammation has clouded the potential influence of other factors released from pyroptotic cells. Here, using a system in which macrophages are induced to undergo pyroptosis without IL-1ß or IL-1α release (denoted Pyro-1), we identify unexpected beneficial effects of the Pyro-1 secretome. First, we noted that the Pyro-1 supernatants upregulated gene signatures linked to migration, cellular proliferation and wound healing. Consistent with this gene signature, Pyro-1 supernatants boosted migration of primary fibroblasts and macrophages, and promoted faster wound closure in vitro and improved tissue repair in vivo. In mechanistic studies, lipidomics and metabolomics of the Pyro-1 supernatants identified the presence of both oxylipins and metabolites, linking them to pro-wound-healing effects. Focusing specifically on the oxylipin prostaglandin E2 (PGE2), we find that its synthesis is induced de novo during pyroptosis, downstream of caspase-1 activation and cyclooxygenase-2 activity; further, PGE2 synthesis occurs late in pyroptosis, with its release dependent on gasdermin D pores opened during pyroptosis. As for the pyroptotic metabolites, they link to immune cell infiltration into the wounds, and polarization to CD301+ macrophages. Collectively, these data advance the concept that the pyroptotic secretome possesses oxylipins and metabolites with tissue repair properties that may be harnessed therapeutically.

TL1A and IL-18 synergy promotes GM-CSF-dependent thymic granulopoiesis in mice.

Acute systemic inflammation critically alters the function of the immune system, often promoting myelopoiesis at the expense of lymphopoiesis. In the thymus, systemic inflammation results in acute thymic atrophy and, consequently, impaired T-lymphopoiesis. The mechanism by which systemic inflammation impacts the thymus beyond suppressing T-cell development is still unclear. Here, we describe how the synergism between TL1A and IL-18 suppresses T-lymphopoiesis to promote thymic myelopoiesis. The protein levels of these two cytokines were elevated in the thymus during viral-induced thymus atrophy infection with murine cytomegalovirus (MCMV) or pneumonia virus of mice (PVM). In vivo administration of TL1A and IL-18 induced acute thymic atrophy, while thymic neutrophils expanded. Fate mapping with Ms4a3-Cre mice demonstrated that thymic neutrophils emerge from thymic granulocyte-monocyte progenitors (GMPs), while Rag1-Cre fate mapping revealed a common developmental path with lymphocytes. These effects could be modeled ex vivo using neonatal thymic organ cultures (NTOCs), where TL1A and IL-18 synergistically enhanced neutrophil production and egress. NOTCH blockade by the LY411575 inhibitor increased the number of neutrophils in the culture, indicating that NOTCH restricted steady-state thymic granulopoiesis. To promote myelopoiesis, TL1A, and IL-18 synergistically increased GM-CSF levels in the NTOC, which was mainly produced by thymic ILC1s. In support, TL1A- and IL-18-induced granulopoiesis was completely prevented in NTOCs derived from Csf2rb-/- mice and by GM-CSFR antibody blockade, revealing that GM-CSF is the essential factor driving thymic granulopoiesis. Taken together, our findings reveal that TL1A and IL-18 synergism induce acute thymus atrophy while  promoting extramedullary thymic granulopoiesis in a NOTCH and GM-CSF-controlled manner.

Virus-Like Particle-Encapsulated Doxorubicin Enters and Kills Murine Tumor Cells Differently from Free Doxorubicin.

The use of nanoparticles as chemotherapeutic carriers has been suggested as a way to overcome a range of side effects associated with classical cancer treatment such as poor selectivity and tumor resurgence. Obtaining precise control of the size and shape of therapeutic nanoparticles is crucial to optimize the targeting of tumor sites. In this work, it is shown that a previously developed system of polypeptide encapsulating individual DNA molecules, that forms rod-shaped nanoparticles of precisely controlled aspect ratio, can be loaded with the DNA-intercalating chemotherapeutic drug doxorubicin (DOX). It is characterized the size and shape of the DOX loaded-Virus-Like DNA Particles (DOX-VLDP) and shown that in this system the DOX payload does not leak out. Through in vitro cell studies, it is shown that DOX-VLDP is internalized by melanoma tumor cells (B16F10 cells) in a delayed and endocytosis-dependent way culminating in increased cytotoxicity and selectivity to tumor cells in comparison with free DOX. In addition, it is found that DOX-VLDP trigger apoptosis and autophagy pathways in treated cells. Taken together, the data on the DOX-VLDP nanoparticles shows that they kill cancer cells differently from free DOX.

Obstetric simulation for undergraduate medical education: how to improve students' self-confidence and expectation according to gender

SUMMARY OBJECTIVE: The aim of this study was to evaluate the effects of obstetric simulation training on undergraduate medical students to improve their self-confidence. METHODS: Fifth-year undergraduate medical students were invited to a 2-week course of simulation in obstetrics during their clerkship. The sessions included were as follows: (1) care for the second and third periods of childbirth, (2) partograph analysis and pelvimetry, (3) premature rupture of membranes at term, and (4) diagnosis and management of third-trimester bleeding. Before the first session and at the end of the training period, a questionnaire about self-confidence in obstetric procedures and skills was applied. RESULTS: A total of 115 medical students were included, of whom 60 (52.2%) were male and 55 (47.8%) were female. Comparing initial and final scores, the median results of the subscales "comprehension and preparation" (18 vs. 22, p<0.001), "knowledge of procedures" (14 vs. 20, p<0.001), and "expectation" (22 vs. 23, p<0.01) were significantly higher at the end of the training period in all items of the questionnaire than in the beginning. Differences were found based on the students' gender, i.e., female students had a significantly higher sum of scores than the male students in the initial subscale for "expectation" (median, 24 vs. 22, p<0.001) and "interest" (median, 23 vs. 21, p=0.032), and a higher sum of scores in the subscale for "expectation" (median, 23 vs. 21, p=0.010) in the final questionnaire. CONCLUSION: Obstetric simulation enhances the improvement of students' self-confidence in understanding both the physiology of childbirth and the obstetric care procedures. Further studies are needed to understand the influence of gender on obstetric care.

Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches.

The liver is the largest solid organ in the body, yet it remains incompletely characterized. Here we present a spatial proteogenomic atlas of the healthy and obese human and murine liver combining single-cell CITE-seq, single-nuclei sequencing, spatial transcriptomics, and spatial proteomics. By integrating these multi-omic datasets, we provide validated strategies to reliably discriminate and localize all hepatic cells, including a population of lipid-associated macrophages (LAMs) at the bile ducts. We then align this atlas across seven species, revealing the conserved program of bona fide Kupffer cells and LAMs. We also uncover the respective spatially resolved cellular niches of these macrophages and the microenvironmental circuits driving their unique transcriptomic identities. We demonstrate that LAMs are induced by local lipid exposure, leading to their induction in steatotic regions of the murine and human liver, while Kupffer cell development crucially depends on their cross-talk with hepatic stellate cells via the evolutionarily conserved ALK1-BMP9/10 axis.

Antibodies directed towards neuraminidase restrict influenza virus replication in primary human bronchial epithelial cells.

Influenza neuraminidase (NA) is implicated in various aspects of the virus replication cycle and therefore is an attractive target for vaccination and antiviral strategies. Here we investigated the potential for NA-specific antibodies to interfere with A(H1N1)pdm09 replication in primary human airway epithelial (HAE) cells. Mouse polyclonal anti-NA sera and a monoclonal antibody could block initial viral entry into HAE cells as well as egress from the cell surface. NA-specific polyclonal serum also reduced virus replication across multiple rounds of infection. Restriction of virus entry correlated with the ability of the serum or monoclonal antibody to mediate neuraminidase inhibition (NI). Finally, human sera with NI activity against the N1 of A(H1N1)pdm09 could decrease H6N1 virus infection of HAE cells, highlighting the potential contribution of anti-NA antibodies in the control of influenza virus infection in humans.

Molecular detection of omicron SARS-CoV-2 variant is achieved by RT-LAMP despite genomic mutations

BACKGROUND Severe acute respiratory syndrome coronavirus (SARS-CoV-2) omicron variant was first detected in South Africa in November 2021. Since then, the number of cases due to this variant increases enormously every day in different parts of the world. Mutations within omicron genome may impair the molecular detection resulting in false negative results during Coronavirus disease 19 (COVID-19) diagnosis. OBJECTIVES To verify if colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) targeting N and E genes would work efficiently to detect omicron SARS-CoV-2 variant and its sub-lineages. METHODS SARS-CoV-2 reverse transcription quantitative polymerase chain reaction (RT-qPCR) positive samples were sequenced by next generation DNA sequencing. The consensus sequences generated were submitted to Pangolin tool for SARS-CoV-2 lineage identification. RT-LAMP reactions were performed at 65ºC/30 min targeting N and E. FINDINGS SARS-CoV-2 omicron can be detected by RT-LAMP targeting N and E genes despite the genomic mutation of this more transmissible lineage. Omicron SARS-CoV-2 sub-lineages were tested and efficiently detected by RT-LAMP. We demonstrated that this test is very sensitive in detecting omicron variant, with LoD as low as 0.4 copies/µL. MAIN CONCLUSIONS Molecular detection of omicron SARS-CoV-2 variant and its sub-lineages can be achieved by RT-LAMP despite the genomic mutations as a very sensitive surveillance tool for COVID-19 molecular diagnosis.

Vivência de cuidadores informais na transição de papéis após o processo de desospitalização / Vivencia de cuidadores informales en la transición de roles después del proceso de desospitalización / Informal caregivers' experience in role transition after dehospitalization

RESUMO Objetivo: apreender as vivências de cuidadores informais de pessoas dependentes no processo de transição de papéis após a desospitalização. Método: estudo descritivo, exploratório, qualitativo, realizado com cuidadores informais de pessoas dependentes que participaram de um protocolo de instrumentalização para alta. A coleta de dados ocorreu com 10 participantes da região sul do país, entre julho e agosto de 2021, por meio de entrevistas conduzidas no domicílio. Para a organização dos dados, utilizou-se o software IRAMUTEQ®. A análise se ancorou na Teoria das Transições. Resultados: os resultados abordaram o processo de transição situacional de cuidadores informais, emergindo três classes finais: Classe 1 - Dificuldades no cotidiano de cuidados com a pessoa dependente; Classe 2 - Significados atribuídos à transição de papéis; e Classe 3 - (Des)Continuidade do cuidado após a alta hospitalar. Considerações finais: pôde-se apreender que as vivências foram permeadas por fatores inibidores, de modo que os significados atribuídos a essa experiência se associaram às dificuldades com a transição para o papel de cuidador. O enfermeiro desempenhou papel de facilitador no processo de transição situacional, ao acompanhá-los, orientá-los e capacitá-los para o desenvolvimento de novas habilidades.

RESUMEN Objetivo: comprender las vivencias de cuidadores informales de personas dependientes en el proceso de transición de roles después de la desospitalización. Método: estudio descriptivo, exploratorio, cualitativo, realizado con cuidadores informales de personas dependientes que participaron de un protocolo de instrumentalización para el alta. La recolección de datos ocurrió con 10 participantes de la región sur del país, entre julio y agosto de 2021, por medio de entrevistas realizadas en el domicilio. Para la organización de los datos se utilizó el software IRAMUTEQ®. El análisis se basó en la Teoría de las Transiciones. Resultados: los resultados trataron el proceso de transición situacional de cuidadores informales, surgiendo tres clases finales: Clase 1 - Dificultades en el cotidiano de cuidados a la persona dependiente; Clase 2 - Significados atribuidos a la transición de roles; y Clase 3 - (Des)Continuidad del cuidado después del alta hospitalaria. Consideraciones finales: se pudo comprender que las vivencias están marcadas por factores inhibidores, de modo que los significados atribuidos a esa experiencia se asociaron a las dificultades con la transición para el rol de cuidador. El enfermero desempeñó un papel de facilitador en el proceso de transición situacional, al acompañarlos, orientarlos y capacitarlos para el desarrollo de nuevas habilidades.

ABSTRACT Objective: to understand the experiences of informal caregivers of dependent people in the role transition process after dehospitalization. Method: a descriptive, exploratory, qualitative study carried out with informal caregivers of dependent people who participated in a protocol of instrumentation for discharge. Data collection took place with 10 participants from southern Brazil, between July and August 2021, through interviews conducted at home. For data organization, we used IRAMUTEQ®. Analysis was anchored in the Transition Theory. Results: the results addressed the process of situational transition of informal caregivers, emerging three final classes: Class 1 - Difficulties in the daily care of dependent people; Class 2 - Meanings given to role transitions; and Class 3 - (Dis)continuity of care after hospital discharge. Final considerations: it was possible to understand that the experiences were permeated by inhibiting factors so that the meanings given to this experience were associated with the difficulties with the transition to the role of caregiver. Nurses played the role of facilitator in the situational transition process, by accompanying them, guiding them and enabling them to develop new skills.

Estresse, má qualidade do sono e desfechos negativos a saúde em estudantes de enfermagem / Stress, poor sleep quality and negative health outcomes in nursing students / Estrés, mala calidad del sueño y resultados negativos de salud en estudiantes de enfermería

RESUMO Objetivo: analisar o perfil biossocial-acadêmico, nível de estresse e qualidade de sono em estudantes de enfermagem no primeiro ano do curso. Método: pesquisa longitudinal, prospectiva e quantitativa, realizada em 2016 junto a estudantes de enfermagem do primeiro ano de duas universidades de São Paulo. Ao início das aulas e ao final do ano letivo, aplicaram-se um formulário biossocial-acadêmico, instrumento para avaliação do estresse em estudantes de enfermagem e índice de qualidade de sono de Pittsburgh. Utilizaram-se os testes de Qui-quadrado e exato de Fischer para comparar o estresse e a qualidade do sono. Resultados: O total de 117 estudantes compuseram a amostra em março e 100 em dezembro de 2016. Evidenciou-se aumento no percentual de estudantes com muito e alto estresse, a duração do sono reduziu significativamente de 6,0 para 5,4 horas em média, por noite, e apresentaram dificuldades no gerenciamento do tempo, formação profissional para seguir o curso e atividades teóricas.Conclusão: o ambiente acadêmico apresenta potencial para o adoecimento do estudante, com alto estresse, má qualidade do sono e desfechos negativos à saúde.

ABSTRACT Objective: to analyze the biosocial-academic profile, level of stress and quality of sleep in nursing students during the first year of the course. Method: longitudinal, prospective and quantitativeresearch, carried out in 2016 with first-year nursing students from two universities in São Paulo. At the beginning of classes and at the end of the school year, the following were applied a biosocial-academic \form, an instrument to assess stress in nursing students and a Pittsburgh sleep quality index. Chi-square and Fisher's exact tests were used to compare stress and sleep quality. Results:117 students comprised the sample in March and 100 in December 2016. There was an increase in the percentage of students with high and high stress, sleep duration significantly reduced from 6.0 to 5.4 hours on average, per night, and had difficulties in time management, professional training to follow the course and theoretical activities.Conclusion:theacademic environment has the potential for the student's illness, with high stress, poor sleep quality and its negative health outcomes.

RESUMEN Objetivo: analizar el perfil biosocial y académico, nivel de estrés y calidad del sueño en estudiantes de enfermería durante el primer año del curso. Método: investigación longitudinal, prospectiva y cuantitativa, realizada en 2016 con estudiantes de primer año de enfermeríade dos universidades de São Paulo. Al inicio de las clases y al final del año lectivo se aplicaron un formulario biosocial y académico, un instrumento para evaluar el estrés en estudiantes de enfermería y un índice de calidad del sueño de Pittsburgh. Paracomparar el estrés y la calidad del sueño se utilizaron las pruebas de chi-cuadrado y exacta de Fisher. Resultados:El total de 117 estudiantes conformaron la muestra en marzo y 100 en diciembre de 2016. Hubo un aumento en el porcentaje de estudiantes conmucho y alto estrés, la duración del sueño se redujo significativamente de 6.0 a 5.4 horas en promedio, por noche, y tuvieron dificultades en la gestión del tiempo. formación profesional para seguir el curso y actividades teóricas. Conclusión:el entorno académico tiene el potencial de que el estudiante se enferme, con alto estrés, mala calidad del sueño y sus resultados negativos para la salud.

Microbes exploit death-induced nutrient release by gut epithelial cells.

Regulated cell death is an integral part of life, and has broad effects on organism development and homeostasis1. Malfunctions within the regulated cell death process, including the clearance of dying cells, can manifest in diverse pathologies throughout various tissues including the gastrointestinal tract2. A long appreciated, yet elusively defined relationship exists between cell death and gastrointestinal pathologies with an underlying microbial component3-6, but the direct effect of dying mammalian cells on bacterial growth is unclear. Here we advance a concept that several Enterobacteriaceae, including patient-derived clinical isolates, have an efficient growth strategy to exploit soluble factors that are released from dying gut epithelial cells. Mammalian nutrients released after caspase-3/7-dependent apoptosis boosts the growth of multiple Enterobacteriaceae and is observed using primary mouse colonic tissue, mouse and human cell lines, several apoptotic triggers, and in conventional as well as germ-free mice in vivo. The mammalian cell death nutrients induce a core transcriptional response in pathogenic Salmonella, and we identify the pyruvate formate-lyase-encoding pflB gene as a key driver of bacterial colonization in three contexts: a foodborne infection model, a TNF- and A20-dependent cell death model, and a chemotherapy-induced mucositis model. These findings introduce a new layer to the complex host-pathogen interaction, in which death-induced nutrient release acts as a source of fuel for intestinal bacteria, with implications for gut inflammation and cytotoxic chemotherapy treatment.

Relações de Amizade, nível de tolerância e fatores associados no contexto do ensino superior / Friendship relationships, tolerance level and associated factors in the context of higher education / Relaciones de amistad, nivel de tolerancia y factores asociados en el contexto de la educación superior

Objetivo: analisar a importância das amizades no processo de tolerância durante o período acadêmico. Método: Estudo transversal, analítico e quantitativo, realizado com 276 discentes da área de saúde de uma instituição privada do estado de Goiás. Foram aplicados formulário sociodemográfico e o instrumento de Avaliação da Tolerância nas Relações de Amizade. A análise deu-se por meio de estatística descritiva e regressão linear, com método backwar. Resultados: As relações interpessoais de amizades facilitam a permanência dos discentes no curso. A religião, o curso de graduação escolhido e o período do curso em que o aluno está matriculado foram fatores que contribuíram para o aumento da tolerância às relações de amizade. Conclusão: Os achados principais sugerem que dois fatores são importantes para a manutenção da tolerância características sociodemográficas e relação interpessoal entre os amigos.

Objective: to assess the importance of friendships in the tolerance process during the academic period. Method: Cross-sectional, analytical and quantitative study, carried out with 276 students in the health area of a private institution in the state of Goiás. A sociodemographic form and the instrument of Assessment of Tolerance in Friendship Relationships were applied. The analysis was carried out through descriptive statistics and linear regression, with the backwar method. Results: Interpersonal friendship relationships facilitate the permanence of students in the course. Religion, the chosen undergraduate course and the period of the course in which the student is enrolled were factors that contributed to the increased tolerance for friendship relationships. Conclusion: The main findings suggest that two factors are important for the maintenance of tolerance, sociodemographic characteristics and interpersonal relationships among friends.

Objetivo: analizar la importancia de las amistades en el proceso de tolerancia durante el período académico. Metodo: estudio transversal, analítico y cuantitativo, realizado con 276 estudiantes del área de la salud de una institución privada del estado de Goiás. Se aplicó un formulario sociodemográfico y el instrumento de Evaluación de la Tolerancia en las Relaciones de Amistad. El análisis se realizó mediante estadística descriptiva y regresión lineal, con el método backwar. Resultados: las relaciones de amistad interpersonal facilitan la permanencia de los estudiantes en el curso. La religión, el curso de graduación elegido y el período del curso en el que está matriculado fueron factores que contribuyeron al aumento de la tolerancia a las relaciones de amistad. Conclusión: los principales hallazgos sugieren que dos factores son importantes para el mantenimiento de la tolerancia, las características sociodemográficas y las relaciones interpersonales entre amigos

A recurrent mutation in tshb gene underlying central congenital hypothyroidism undetectable in neonatal screening / Uma mutação recorrente no gene tshb resultando em hipotireoidismo congênito central não detectável na triagem neonatal

ABSTRACT Objective: To describe the case of a patient with central congenital hypothyroidism (CCH) due to a recurrent mutation in the TSHB gene, as well as to conduct a genetic study of his family. Case description: It is presented a case report of a 5-month-old boy with a delayed diagnosis of isolated CCH in whom the molecular analysis was performed 12 years later and detected a recurrent mutation (c.373delT) in TSHB gene. The parents and sister were carriers of the mutant allele. Comments: The c.373delT mutation has previously been reported in patients from Brazil, Germany, Belgium, United States, Switzerland, Argentina, France, Portugal, United Kingdom and Ireland. In summary, our case and other ones reported in the literature support the theory that this mutation may be a common cause of isolated TSH deficiency. Isolated TSH deficiency is not detected by routine TSH-based neonatal screening, representing a clinical challenge. Therefore, when possible, molecular genetic study is indicated. Identification of affected and carriers allows the diagnosis, treatment and adequate genetic counseling.

RESUMO Objetivo: Descrever o caso de um paciente com hipotireoidismo congênito central (HCC) por conta de uma mutação recorrente no gene TSHB, bem como realizar um estudo genético de sua família. Descrição do caso: Relato de caso de um menino de 5 meses de idade com diagnóstico tardio de HCC isolado, em quem a análise molecular foi realizada 12 anos depois e detectou uma mutação recorrente (c.373delT) no gene TSHB. Os pais e a irmã eram portadores do alelo mutante. Comentários: A mutação c.373delT já foi relatada em pacientes do Brasil, da Alemanha, da Bélgica, dos Estados Uinidos, da Suíça, da Argentina, da França, de Portugal, do Reino Unido e da Irlanda. Em resumo, nosso caso e outros relatados na literatura reforçam a teoria de que essa mutação pode ser uma causa comum de deficiência isolada de TSH. A deficiência isolada de TSH não é detectada na triagem neonatal com base na dosagem de TSH, representando um desafio clínico. Portanto, quando possível, o estudo genético molecular é indicado. A identificação dos afetados e dos portadores permite o diagnóstico, o tratamento e o aconselhamento genético adequado.

Epithelial HMGB1 Delays Skin Wound Healing and Drives Tumor Initiation by Priming Neutrophils for NET Formation.

Regenerative responses predispose tissues to tumor formation by largely unknown mechanisms. High-mobility group box 1 (HMGB1) is a danger-associated molecular pattern contributing to inflammatory pathologies. We show that HMGB1 derived from keratinocytes, but not myeloid cells, delays cutaneous wound healing and drives tumor formation. In wounds of mice lacking HMGB1 selectively in keratinocytes, a marked reduction in neutrophil extracellular trap (NET) formation is observed. Pharmacological targeting of HMGB1 or NETs prevents skin tumorigenesis and accelerates wound regeneration. HMGB1-dependent NET formation and skin tumorigenesis is orchestrated by tumor necrosis factor (TNF) and requires RIPK1 kinase activity. NETs are present in the microenvironment of keratinocyte-derived tumors in mice and lesional and tumor skin of patients suffering from recessive dystrophic epidermolysis bullosa, a disease in which skin blistering predisposes to tumorigenesis. We conclude that tumorigenicity of the wound microenvironment depends on epithelial-derived HMGB1 regulating NET formation, thereby establishing a mechanism linking reparative inflammation to tumor initiation.

Blocking connexin43 hemichannels protects mice against tumour necrosis factor-induced inflammatory shock.

Upon intravenous injection of tumour necrosis factor (TNF) in mice, a systemic inflammatory response syndrome (SIRS) is initiated, characterized by an acute cytokine storm and induction of vascular hyperpermeability. Connexin43 hemichannels have been implicated in various pathological conditions, e.g. ischemia and inflammation, and can lead to detrimental cellular outcomes. Here, we explored whether targeting connexin43 hemichannels could alleviate TNF-induced endothelial barrier dysfunction and lethality in SIRS. Therefore, we verified whether administration of connexin43-targeting-peptides affected survival, body temperature and vascular permeability in vivo. In vitro, TNF-effects on connexin43 hemichannel function were investigated by single-channel studies and Ca2+-imaging. Blocking connexin43 hemichannels with TAT-Gap19 protected mice against TNF-induced mortality, hypothermia and vascular leakage, while enhancing connexin43 hemichannel function with TAT-CT9 provoked opposite sensitizing effects. In vitro patch-clamp studies revealed that TNF acutely activated connexin43 hemichannel opening in endothelial cells, which was promoted by CT9, and inhibited by Gap19 and intracellular Ca2+-buffering. In vivo experiments aimed at buffering intracellular Ca2+, and pharmacologically targeting Ca2+/calmodulin-dependent protein kinase-II, a known modulator of endothelial barrier integrity, demonstrated their involvement in permeability alterations. Our results demonstrate significant benefits of inhibiting connexin43 hemichannels to counteract TNF-induced SIRS-associated vascular permeability and lethality.

Stromal-targeting radioimmunotherapy mitigates the progression of therapy-resistant tumors.

Radioimmunotherapy (RIT) aims to deliver a high radiation dose to cancer cells, while minimizing the exposure of normal cells. Typically, monoclonal antibodies are used to target the radionuclides to cancer cell surface antigens. However, antibodies face limitations due to their poor tumor penetration and suboptimal pharmacokinetics, while the expression of their target on the cancer cell surface may be gradually lost. In addition, most antigens are expressed in a limited number of tumor types. To circumvent these problems, we developed a Nanobody (Nb)-based RIT against a prominent stromal cell (stromal-targeting radioimmunotherapy or STRIT) present in nearly all tumors, the tumor-associated macrophage (TAM). Macrophage Mannose Receptor (MMR) functions as a stable molecular target on TAM residing in hypoxic areas, further allowing the delivery of a high radiation dose to the more radioresistant hypoxic tumor regions. Since MMR expression is not restricted to TAM, we first optimized a strategy to block extra-tumoral MMR to prevent therapy-induced toxicity. A 100-fold molar excess of unlabeled bivalent Nb largely blocks extra-tumoral binding of 177Lu-labeled anti-MMR Nb and prevents toxicity, while still allowing the intra-tumoral binding of the monovalent Nb. Interestingly, three doses of 177Lu-labeled anti-MMR Nb resulted in a significantly retarded tumor growth, thereby outcompeting the effects of anti-PD1, anti-VEGFR2, doxorubicin and paclitaxel in the TS/A mammary carcinoma model. Together, these data propose anti-MMR STRIT as a valid new approach for cancer treatment.

A RECURRENT MUTATION IN TSHB GENE UNDERLYING CENTRAL CONGENITAL HYPOTHYROIDISM UNDETECTABLE IN NEONATAL SCREENING.

OBJECTIVE: To describe the case of a patient with central congenital hypothyroidism (CCH) due to a recurrent mutation in the TSHB gene, as well as to conduct a genetic study of his family. CASE DESCRIPTION: It is presented a case report of a 5-month-old boy with a delayed diagnosis of isolated CCH in whom the molecular analysis was performed 12 years later and detected a recurrent mutation (c.373delT) in TSHB gene. The parents and sister were carriers of the mutant allele. COMMENTS: The c.373delT mutation has previously been reported in patients from Brazil, Germany, Belgium, United States, Switzerland, Argentina, France, Portugal, United Kingdom and Ireland. In summary, our case and other ones reported in the literature support the theory that this mutation may be a common cause of isolated TSH deficiency. Isolated TSH deficiency is not detected by routine TSH-based neonatal screening, representing a clinical challenge. Therefore, when possible, molecular genetic study is indicated. Identification of affected and carriers allows the diagnosis, treatment and adequate genetic counseling.

Protein crystallization promotes type 2 immunity and is reversible by antibody treatment.

Although spontaneous protein crystallization is a rare event in vivo, Charcot-Leyden crystals (CLCs) consisting of galectin-10 (Gal10) protein are frequently observed in eosinophilic diseases, such as asthma. We found that CLCs derived from patients showed crystal packing and Gal10 structure identical to those of Gal10 crystals grown in vitro. When administered to the airways, crystalline Gal10 stimulated innate and adaptive immunity and acted as a type 2 adjuvant. By contrast, a soluble Gal10 mutein was inert. Antibodies directed against key epitopes of the CLC crystallization interface dissolved preexisting CLCs in patient-derived mucus within hours and reversed crystal-driven inflammation, goblet-cell metaplasia, immunoglobulin E (IgE) synthesis, and bronchial hyperreactivity (BHR) in a humanized mouse model of asthma. Thus, protein crystals may promote hallmark features of asthma and are targetable by crystal-dissolving antibodies.

Damage on plants activates Ca2+-dependent metacaspases for release of immunomodulatory peptides.

Physical damage to cells leads to the release of immunomodulatory peptides to elicit a wound defense response in the surrounding tissue. In Arabidopsis thaliana, the plant elicitor peptide 1 (Pep1) is processed from its protein precursor, PRECURSOR OF PEP1 (PROPEP1). We demonstrate that upon damage, both at the tissue and single-cell levels, the cysteine protease METACASPASE4 (MC4) is instantly and spatiotemporally activated by binding high levels of Ca2+ and is necessary and sufficient for Pep1 maturation. Cytosol-localized PROPEP1 and MC4 react only after loss of plasma membrane integrity and prolonged extracellular Ca2+ entry. Our results reveal that a robust mechanism consisting of conserved molecular components links the intracellular and Ca2+-dependent activation of a specific cysteine protease with the maturation of damage-induced wound defense signals.

DPP8/DPP9 inhibition elicits canonical Nlrp1b inflammasome hallmarks in murine macrophages.

Activating germline mutations in the human inflammasome sensor NLRP1 causes palmoplantar dyskeratosis and susceptibility to Mendelian autoinflammatory diseases. Recent studies have shown that the cytosolic serine dipeptidyl peptidases DPP8 and DPP9 suppress inflammasome activation upstream of NLRP1 and CARD8 in human keratinocytes and peripheral blood mononuclear cells. Moreover, pharmacological inhibition of DPP8/DPP9 protease activity was shown to induce pyroptosis in murine C57BL/6 macrophages without eliciting other inflammasome hallmark responses. Here, we show that DPP8/DPP9 inhibition in macrophages that express a Bacillus anthracis lethal toxin (LeTx)-sensitive Nlrp1b allele triggered significantly accelerated pyroptosis concomitant with caspase-1 maturation, ASC speck assembly, and secretion of mature IL-1ß and IL-18. Genetic ablation of ASC prevented DPP8/DPP9 inhibition-induced caspase-1 maturation and partially hampered pyroptosis and inflammasome-dependent cytokine release, whereas deletion of caspase-1 or gasdermin D triggered apoptosis in the absence of IL-1ß and IL-18 secretion. In conclusion, blockade of DPP8/DPP9 protease activity triggers rapid pyroptosis and canonical inflammasome hallmarks in primary macrophages that express a LeTx-responsive Nlrp1b allele.