Systematization of initiatives in sexual and reproductive health about good practices criteria in response to the COVID-19 pandemic in primary health care in Chile.

Introduction The COVID- 19 pandemic discontinued sexual and reproductive health care in Chile and the world. The national focus on hospital care led primary care teams to respond in natural and diverse ways. Understanding the factors involved in this process may improve future responses from the judgment of good practices. Therefore, this study aimed to identify and systematize sexual and reproductive health initiatives raised by primary care teams in response to the COVID- 19 pandemic in Chile. Methods We systematically evaluated initiatives and practices in sexual and reproductive health in prima-ry care between June 2020 and November 2021. This study was developed in three methodological phases: a review of documents, a collection of experiences through an electronic instrument sent to the 29 health services in Chile, and in-depth interviews. According to best practice criteria, mapping and characterizing the initiatives and critical discourse analysis of narratives and interviews were carried out. Results Forty-one initiatives from 19 health services were identified, mainly from the South Central macro zone and urban areas. In these areas, care was recognized. These practices were relevant, aligned with their objectives, rapidly implemented, and used novel strategies through new technologies. However, these initiatives had little intercultural relevance or evaluation. Perceived success was related to motivation, leadership, and institutional and community resilience. The adaptability of initiatives emerged as a new need and criterion of analysis. Conclusion The lessons learned from these initiatives invite us to consider health care teams' mental health, their relationship with the community, the use of new technologies, the evaluation of practices considering satisfaction, cross- cutting approaches, and their adaptability. In all, these aspects may improve primary care response in sexual and reproductive health to new crises.

Introducción La pandemia de COVID- 19 ha implicado la discontinuidad de atención en salud sexual y reproductiva en Chile y el mundo. El foco en la contención hospitalaria de la pandemia llevó a los equipos de atención primaria a responder de manera innata y diversa. Por lo tanto, el objetivo de este estudio fue identificar y sistematizar iniciativas de salud sexual y reproductiva planteadas por equipos de atención primaria en respuesta a la pandemia COVID- 19 en Chile, con el fin de comprender y aprender de los factores involucrados en esta, para mejorar futuras respuestas desde la lógica de buenas prácticas. Metodología Estudio de sistematización de iniciativas y prácticas en salud sexual y reproductiva en atención primaria, entre junio de 2020 y noviembre de 2021. Fue desarrollado en tres fases metodológicas: revisión de documentos, recolección de experiencias a través de instrumento electrónico enviado a los 29 servicios de salud de Chile y entrevistas de profundización. Se realizó un mapeo y caracterización de las iniciativas y análisis crítico de discurso de narrativas y entrevistas, según criterios de buenas prácticas. Resultados Se identificaron 41 iniciativas de 19 servicios de salud, principalmente de la Macro Zona Centro- sur, urbanas y de reorganización de atención. Fueron prácticas pertinentes, alineadas a sus objetivos, con procesos rápidos de implementación, estrategias innovadoras, alto uso de tecnologías con escasa pertinencia intercultural ni evaluación. El éxito percibido se relacionó con motivación, liderazgo, y resiliencia institucional y comunitaria. La adaptabilidad de las iniciativas emergió como una nueva necesidad y criterio. Conclusión Son importantes los aprendizajes emanados de las iniciativas que invitan a considerar la salud mental de los equipos, su relación con la comunidad, el uso de tecnologías, la evaluación de las prácticas considerando satisfacción, enfoques transversales y la adaptabilidad de estas, para mejorar la respuesta de atención primaria en salud sexual y reproductiva ante nuevas crisis.

Sistematización de iniciativas en salud sexual y reproductiva según criterios de buenas prácticas en repuesta a la pandemia COVID-19 en la atención primaria en Chile / Systematization of initiatives in sexual and reproductive health about good practices criteria in response to the COVID-19 pandemic in primary health care in Chile

Introducción La pandemia de COVID- 19 ha implicado la discontinuidad de atención en salud sexual y reproductiva en Chile y el mundo. El foco en la contención hospitalaria de la pandemia llevó a los equipos de atención primaria a responder de manera innata y diversa. Por lo tanto, el objetivo de este estudio fue identificar y sistematizar iniciativas de salud sexual y reproductiva planteadas por equipos de atención primaria en respuesta a la pandemia COVID- 19 en Chile, con el fin de comprender y aprender de los factores involucrados en esta, para mejorar futuras respuestas desde la lógica de buenas prácticas. Metodología Estudio de sistematización de iniciativas y prácticas en salud sexual y reproductiva en atención primaria, entre junio de 2020 y noviembre de 2021. Fue desarrollado en tres fases metodológicas: revisión de documentos, recolección de experiencias a través de instrumento electrónico enviado a los 29 servicios de salud de Chile y entrevistas de profundización. Se realizó un mapeo y caracterización de las iniciativas y análisis crítico de discurso de narrativas y entrevistas, según criterios de buenas prácticas. Resultados Se identificaron 41 iniciativas de 19 servicios de salud, principalmente de la Macro Zona Centro- sur, urbanas y de reorganización de atención. Fueron prácticas pertinentes, alineadas a sus objetivos, con procesos rápidos de implementación, estrategias innovadoras, alto uso de tecnologías con escasa pertinencia intercultural ni evaluación. El éxito percibido se relacionó con motivación, liderazgo, y resiliencia institucional y comunitaria. La adaptabilidad de las iniciativas emergió como una nueva necesidad y criterio. Conclusión Son importantes los aprendizajes emanados de las iniciativas que invitan a considerar la salud mental de los equipos, su relación con la comunidad, el uso de tecnologías, la evaluación de las prácticas considerando satisfacción, enfoques transversales y la adaptabilidad de estas, para mejorar la respuesta de atención primaria en salud sexual y reproductiva ante nuevas crisis.

Introducción La pandemia de COVID- 19 ha implicado la discontinuidad de atención en salud sexual y reproductiva en Chile y el mundo. El foco en la contención hospitalaria de la pandemia llevó a los equipos de atención primaria a responder de manera innata y diversa. Por lo tanto, el objetivo de este estudio fue identificar y sistematizar iniciativas de salud sexual y reproductiva planteadas por equipos de atención primaria en respuesta a la pandemia COVID- 19 en Chile, con el fin de comprender y aprender de los factores involucrados en esta, para mejorar futuras respuestas desde la lógica de buenas prácticas. Metodología Estudio de sistematización de iniciativas y prácticas en salud sexual y reproductiva en atención primaria, entre junio de 2020 y noviembre de 2021. Fue desarrollado en tres fases metodológicas: revisión de documentos, recolección de experiencias a través de instrumento electrónico enviado a los 29 servicios de salud de Chile y entrevistas de profundización. Se realizó un mapeo y caracterización de las iniciativas y análisis crítico de discurso de narrativas y entrevistas, según criterios de buenas prácticas. Resultados Se identificaron 41 iniciativas de 19 servicios de salud, principalmente de la Macro Zona Centro- sur, urbanas y de reorganización de atención. Fueron prácticas pertinentes, alineadas a sus objetivos, con procesos rápidos de implementación, estrategias innovadoras, alto uso de tecnologías con escasa pertinencia intercultural ni evaluación. El éxito percibido se relacionó con motivación, liderazgo, y resiliencia institucional y comunitaria. La adaptabilidad de las iniciativas emergió como una nueva necesidad y criterio. Conclusión Son importantes los aprendizajes emanados de las iniciativas que invitan a considerar la salud mental de los equipos, su relación con la comunidad, el uso de tecnologías, la evaluación de las prácticas considerando satisfacción, enfoques transversales y la adaptabilidad de estas, para mejorar la respuesta de atención primaria en salud sexual y reproductiva ante nuevas crisis.

Development of a small panel of SNPs to infer ancestry in Chileans that distinguishes Aymara and Mapuche components.

BACKGROUND: Current South American populations trace their origins mainly to three continental ancestries, i.e. European, Amerindian and African. Individual variation in relative proportions of each of these ancestries may be confounded with socio-economic factors due to population stratification. Therefore, ancestry is a potential confounder variable that should be considered in epidemiologic studies and in public health plans. However, there are few studies that have assessed the ancestry of the current admixed Chilean population. This is partly due to the high cost of genome-scale technologies commonly used to estimate ancestry. In this study we have designed a small panel of SNPs to accurately assess ancestry in the largest sampling to date of the Chilean mestizo population (n = 3349) from eight cities. Our panel is also able to distinguish between the two main Amerindian components of Chileans: Aymara from the north and Mapuche from the south. RESULTS: A panel of 150 ancestry-informative markers (AIMs) of SNP type was selected to maximize ancestry informativeness and genome coverage. Of these, 147 were successfully genotyped by KASPar assays in 2843 samples, with an average missing rate of 0.012, and a 0.95 concordance with microarray data. The ancestries estimated with the panel of AIMs had relative high correlations (0.88 for European, 0.91 for Amerindian, 0.70 for Aymara, and 0.68 for Mapuche components) with those obtained with AXIOM LAT1 array. The country's average ancestry was 0.53 ± 0.14 European, 0.04 ± 0.04 African, and 0.42 ± 0.14 Amerindian, disaggregated into 0.18 ± 0.15 Aymara and 0.25 ± 0.13 Mapuche. However, Mapuche ancestry was highest in the south (40.03%) and Aymara in the north (35.61%) as expected from the historical location of these ethnic groups. We make our results available through an online app and demonstrate how it can be used to adjust for ancestry when testing association between incidence of a disease and nongenetic risk factors. CONCLUSIONS: We have conducted the most extensive sampling, across many different cities, of current Chilean population. Ancestry varied significantly by latitude and human development. The panel of AIMs is available to the community for estimating ancestry at low cost in Chileans and other populations with similar ancestry.

Neutrophil extracellular traps impair fungal clearance in a mouse model of invasive pulmonary aspergillosis.

Neutrophil extracellular traps (NETs) are formed by polymorphonuclear neutrophils (PMN) and contribute to the innate host defense by binding and killing bacterial and fungal pathogens. Because NET formation depends on histone hypercitrullination by peptidylarginine deiminase 4 (PAD4), we used PAD4 gene deficient (Pad4-/-) mice in a mouse model of invasive pulmonary aspergillosis (IPA) to address the contribution of NETs to the innate host defense in vivo. After the induction (24 h) of IPA by i.t. infection with Aspergillus fumigatus conidia, Pad4-/- mice revealed lower fungal burden in the lungs, accompanied by less acute lung injury, TNFα and citH3 compared to wildtype controls. These findings suggest that release of NETs contributes to tissue damage and limits control of fungal outgrowth. Thus inhibition of NETosis might be a useful strategy to maintain neutrophil function and avoid lung damage in patients suffering from IPA, especially in those suffering from preexisting pulmonary disease.

Development of a small panel of SNPs to infer ancestry in Chileans that distinguishes Aymara and Mapuche components

BACKGROUND: Current South American populations trace their origins mainly to three continental ancestries, i.e. European, Amerindian and African. Individual variation in relative proportions of each of these ancestries may be confounded with socio-economic factors due to population stratification. Therefore, ancestry is a potential confounder variable that should be considered in epidemiologic studies and in public health plans. However, there are few studies that have assessed the ancestry of the current admixed Chilean population. This is partly due to the high cost of genome-scale technologies commonly used to estimate ancestry. In this study we have designed a small panel of SNPs to accurately assess ancestry in the largest sampling to date of the Chilean mestizo population (n = 3349) from eight cities. Our panel is also able to distinguish between the two main Amerindian components of Chileans: Aymara from the north and Mapuche from the south. RESULTS: A panel of 150 ancestry-informative markers (AIMs) of SNP type was selected to maximize ancestry informativeness and genome coverage. Of these, 147 were successfully genotyped by KASPar assays in 2843 samples, with an average missing rate of 0.012, and a 0.95 concordance with microarray data. The ancestries estimated with the panel of AIMs had relative high correlations (0.88 for European, 0.91 for Amerindian, 0.70 for Aymara, and 0.68 for Mapuche components) with those obtained with AXIOM LAT1 array. The country's average ancestry was 0.53 ± 0.14 European, 0.04 ± 0.04 African, and 0.42 ± 0.14 Amerindian, disaggregated into 0.18 ± 0.15 Aymara and 0.25 ± 0.13 Mapuche. However, Mapuche ancestry was highest in the south (40.03%) and Aymara in the north (35.61%) as expected from the historical location of these ethnic groups. We make our results available through an online app and demonstrate how it can be used to adjust for ancestry when testing association between incidence of a disease and nongenetic risk factors. CONCLUSIONS: We have conducted the most extensive sampling, across many different cities, of current Chilean population. Ancestry varied significantly by latitude and human development. The panel of AIMs is available to the community for estimating ancestry at low cost in Chileans and other populations with similar ancestry.

Transcutaneous immunization with CD40 ligation boosts cytotoxic T lymphocyte mediated antitumor immunity independent of CD4 helper cells in mice.

Transcutaneous immunization (TCI) is a novel vaccination strategy that utilizes skin-associated lymphatic tissue to induce immune responses. Employing T-cell epitopes and the TLR7 agonist imiquimod onto intact skin mounts strong primary, but limited memory CTL responses. To overcome this limitation, we developed a novel imiquimod-containing vaccination platform (IMI-Sol) rendering superior primary CD8+ and CD4+ T-cell responses. However, it has been unclear whether IMI-Sol per se is restricted in terms of memory formation and tumor protection. In our present work, we demonstrate that the combined administration of IMI-Sol and CD40 ligation unleashes fullblown specific T-cell responses in the priming and memory phase, strongly enhancing antitumor protection in mice. Interestingly, these effects were entirely CD4+ T cell independent, bypassing the necessity of helper T cells. Moreover, blockade of CD70 in vivo abrogated the boosting effect of CD40 ligation, indicating that the adjuvant effect of CD40 in TCI is mediated via CD70 on professional APCs. Furthermore, this work highlights the so far underappreciated importance of the CD70/CD27 interaction as a promising adjuvant target in TCI. Summing up, we demonstrate that the novel formulation IMI-Sol represents a powerful vaccination platform when applied in combination with sufficient adjuvant thereby overcoming current limitations of TCI.

Antifungal Drugs Influence Neutrophil Effector Functions.

There is a growing body of evidence for immunomodulatory side effects of antifungal agents on different immune cells, e.g., T cells. Therefore, the aim of our study was to clarify these interactions with regard to the effector functions of polymorphonuclear neutrophils (PMN). Human PMN were preincubated with fluconazole (FLC), voriconazole (VRC), posaconazole (POS), isavuconazole (ISA), caspofungin (CAS), micafungin (MFG), conventional amphotericin B (AMB), and liposomal amphotericin B (LAMB). PMN then were analyzed by flow cytometry for activation, degranulation, and phagocytosis and by dichlorofluorescein assay to detect reactive oxygen species (ROS). Additionally, interleukin-8 (IL-8) release was measured by enzyme-linked immunosorbent assay. POS led to enhanced activation, degranulation, and generation of ROS, whereas IL-8 release was reduced. In contrast, ISA-pretreated PMN showed decreased activation signaling, impaired degranulation, and lower generation of ROS. MFG caused enhanced expression of activation markers but impaired degranulation, phagocytosis, generation of ROS, and IL-8 release. CAS showed increased phagocytosis, whereas degranulation and generation of ROS were reduced. AMB led to activation of almost all effector functions besides impaired phagocytosis, whereas LAMB did not alter any effector functions. Independent from class, antifungal agents show variable influence on neutrophil effector functions in vitro Whether this is clinically relevant needs to be clarified.

Clinical features and possible founder mutation of the 8bp duplication mutation in the SLC4A11 gene causing corneal dystrophy and perceptive deafness in three South American families.

BACKGROUND: Corneal Dystrophy and Perceptive Deafness (CDPD) or Harboyan syndrome is an autosomal recessive rare disorder, characterized by congenital corneal opacities and progressive sensorineural hearing loss, which usually begins after the second decades of life. This study reports the ophthalmic, audiological and genetic features, in five CDPD affected patients from three Chilean families. MATERIALS AND METHODS: Five individuals affected with CDPD from three unrelated Chilean families were clinically and genetically examined. To evaluate a putative founder mutation 7 SNPs were analyzed in the three families, an Argentinian patient (carrier of the same mutation previously reported) and 87 Chilean controls. RESULTS: The ophthalmic symptoms in the five patients were bilateral and symmetric, starting before one year of age, and visual acuity varied from 0.1 to 0.3. In all cases, hearing loss began over 8 years old. The sequence of the 19 exons of SLC4A11 gene of all the affected patients exhibited homozygous eight nucleotide sequence duplication (c.2233_2240dup TATGACAC, p.(Ile748Metfs*5)) at the end of exon 16. All the affected patients of the three families were homozygous for a haplotype composed of five SNPs and covering 4,1 Mb. The same haplotype was present in one allele of the heterozygous Argentinean patient and has a frequency of 2.76% in Chilean population. CONCLUSIONS: The five CDPD patients were homozygous for the same mutation in the SLC4A11 gene. Haplotype analysis of all the affected, including the case reported from Argentina was in accordance with a founder mutation.

Tumor immunoevasion via acidosis-dependent induction of regulatory tumor-associated macrophages.

Many tumors evolve sophisticated strategies to evade the immune system, and these represent major obstacles for efficient antitumor immune responses. Here we explored a molecular mechanism of metabolic communication deployed by highly glycolytic tumors for immunoevasion. In contrast to colon adenocarcinomas, melanomas showed comparatively high glycolytic activity, which resulted in high acidification of the tumor microenvironment. This tumor acidosis induced Gprotein-coupled receptor-dependent expression of the transcriptional repressor ICER in tumor-associated macrophages that led to their functional polarization toward a non-inflammatory phenotype and promoted tumor growth. Collectively, our findings identify a molecular mechanism of metabolic communication between non-lymphoid tissue and the immune system that was exploited by high-glycolytic-rate tumors for evasion of the immune system.

Author Correction: ADAMTS-13 regulates neutrophil recruitment in a mouse model of invasive pulmonary aspergillosis.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Idelalisib impairs TREM-1 mediated neutrophil inflammatory responses.

Triggering receptor expressed on myeloid cells (TREM)-1 on polymorphonuclear neutrophils (PMN) regulates innate immune activation in infectious and non-infectious conditions. TREM-1 ligation activates phosphatidyl-inositol 3 kinase (PI3K) triggering all neutrophil effector functions. As idelalisib is a PI3K inhibitor in clinical use for the treatment of non-Hodgkin lymphomas, we asked whether this inhibitor affects PMN functionalities. We analyzed PMNs from healthy donors or lymphoma patients for oxidative burst, phagocytosis, activation markers and IL-8 release upon TREM-1 or TLR ligation ex vivo. In addition, we performed western blot analyses to characterize the signaling events inhibited by idelalisib and other PI3K inhibitors. Upon TREM-1 ligation, the oxidative burst, degranulation, L-selectin shedding and cytokine release were all strongly reduced in the presence of idelalisib along impaired phosphorylation of P38, AKT and ERK by western blot analyses. In line with this, PMNs from patients receiving idelalisib also displayed an impaired TREM-1 mediated PMN activation ex vivo. In conclusion, PI3K inhibitors might cause a neutropenia-like susceptibility to infections in patients by leading to impaired PMN functionality. This should be considered when evaluating patients for infections treated with such inhibitors in daily clinical routine.

¿Qué opinan los residentes sobre sus profesores? Un enfoque cualitativo / What do residents think about their tutors? A qualitative approach

INTRODUCCIÓN: La interacción de los profesores con los estudiantes, la supervisión de las prácticas clínicas y el profesionalismo son temas que explora el instrumento de evaluación del desempeño docente que recoge la opinión de los residentes sobre las competencias educativas de profesores titulares y adjuntos. OBJETIVO: 1)Recuperar los testimonios de los residentes sobre el modelo de rol y el compromiso de sus profesores con su aprendizaje; 2)reconocer las estrategias educativas empleadas por los docentes en contextos clínicos; 3)visibilizar las dificultades de contratación en la planta académica actual del PUEM que afectan el proceso educativo, y 4)reportar las menciones de maltrato y percepción de injusticia de los residentes por sus profesores. MATERIAL Y MÉTODO: Estudio cualitativo que consideró los comentarios a pregunta abierta de 1.628 residentes sobre el desempeño docente en las residencias médicas. Se clasificaron en 10 categorías con subcategorías y se cuantificaron las tendencias positiva y negativa. Posteriormente se analizaron los testimonios de acuerdo a los 3 temas del cuestionario. RESULTADOS: Para los residentes, las categorías del profesor como modelo a seguir y el compromiso académico del docente obtuvieron el mayor número de opiniones positivas, mientras que la contratación del profesor y las estrategias educativas empleadas por este último reportaron el mayor número de comentarios negativos. DISCUSIÓN: Los comentarios libres que plasmaron los residentes sobre el desempeño docente reflejan las fortalezas y debilidades del proceso educativo en contextos hospitalarios, lo que permite un acercamiento cualitativo a la relación con sus profesores

INTRODUCTION: Teacher and student interaction, the monitoring of clinical practices, and professionalism are the issues that the teaching performance assessment tool examines by gathering the opinions of the residents on the educational skills of teaching staff and lecturers. OBJECTIVE: The aim of this study was to: (I)gathering the comments of the residents about the role model and commitment of tutors to their training; (II)to determine the educational strategies used by teachers in clinical settings; (III)to show the current recruitment difficulties of the Medical Specialisations Plan (PUEM) affect the educational process, and (IV)to report the residents mentions of abuse and perceived unfairness by their teachers. MATERIAL AND METHOD: Qualitative study that considered the written comments by 1,628 residents in an open question regarding teaching performance in medical residencies. They were classified into 10 categories with sub-categories and the positive and negative trends were quantified. The comments were analysed according to the 3 topics of the questionnaire. RESULTS: For residents, the categories of the educator as role model and academic commitment of the teacher obtained the largest number of positive reviews, while hiring of teachers and educational strategies employed by the latter reported the highest number of negative comments. DISCUSSION: The free comments expressed by the residents on the teaching performance reflect the strengths and weaknesses of the teaching process in hospital contexts, which provides a qualitative approach to the relationship with their teachers

ADAMTS-13 regulates neutrophil recruitment in a mouse model of invasive pulmonary aspergillosis.

Von Willebrand factor (VWF) is secreted as an acute phase protein during inflammation. ADAMTS-13 regulates the size and prothrombotic activity of VWF by it's specific proteolytic activity. To determine the relevance of this regulatory pathway for the innate inflammatory response by polymorphonuclear neutrophils (PMN), we employed a mouse model of invasive pulmonary aspergillosis (IPA) where PMN functionality is crucial for fungal clearance and survival. IPA was induced by intratracheal application of Aspergillus fumigatus (A. fumigatus) conidia in wildtype (129/Sv/Pas) or ADAMTS-13 deficient (Adamts13 -/-) mice. While neutropenic mice developed lethal IPA, all wildtype mice survived the infection. In contrast to wildtype or VWF deficient mice, Adamts13 -/- mice displayed more severe signs of disease with a lethal course in 24% with an increased fungal burden and signs of acute lung injury. Histology sections demonstrated a more pronounced perivascular leukocyte infiltration in support of a dysregulated inflammatory response in Adamts13 -/- mice. Importantly, we observed no general defect in the activation of neutrophil functions in response to conidia or hyphae in vitro. Therefore, we conclude that the proteolytic regulation of VWF by ADAMTS-13 or ADAMTS-13 by itself is an important mechanism to control PMN recruitment in acute inflammatory processes, such as fungal pneumonias.

9-Phenanthrol enhances the generation of an CD8+ T cell response following transcutaneous immunization with imiquimod in mice.

BACKGROUND: Transcutaneous immunization (TCI) is a non-invasive vaccination strategy targeting the skin-associated lymphoid tissue. Topical application of the TLR7 agonist imiquimod as adjuvant in combination with peptide antigens activates the innate immune system and mounts cytotoxic T lymphocyte (CTL) responses. OBJECTIVE: Based on the commercial 5% imiquimod-containing drug Aldara we aimed to develop an improved formulation with superior vaccination efficiencies. The primary target was the enhancement of mast cell activation as important key for the function of the innate immune system. METHODS: We investigated the effects of 9-phenanthrol (9-phe) on the activation of mast cells in vitro and in vivo. For TCI, we applied 0.2% 9-phe in Aldara or Aldara alone as adjuvants in combination with the MHC class I - restricted peptide SIINFEKL. To monitor vaccination, mast cell degranulation, migration of DC and frequencies of epitope-specific CTL was assessed. In a transgenic tumor model, the efficiencies of prophylactic immunization against a tumor antigen were also monitored. RESULTS: 9-phe induced degranulation of mast cells in vitro and upon topical application in vivo. A mixture of 0.2% 9-phe in Aldara showed superior results regarding the migration of DC and the expansion of antigen-specific CTL. Consequently, prophylactic immunization with 0.2% 9-phe in Aldara caused enhanced protection against tumor inoculation. CONCLUSION: Our data demonstrate that a simple modification of an adjuvant formulation can yield superior results in experimental vaccination protocols by boosting critical steps leading to the generation of an efficient CTL response.

Combined immunotherapy: CTLA-4 blockade potentiates anti-tumor response induced by transcutaneous immunization.

BACKGROUND: The epidermal application of the Toll Like Receptor 7 agonist imiquimod and a T-cell peptide epitope (transcutaneous immunization, TCI) mediates systemic peptide-specific cytotoxic T-cell (CTL) responses and leads to tumor protection in a prophylactic tumor setting. However, it does not accomplish memory formation or permanent defiance of tumors in a therapeutic set-up. As a distinct immunologic approach, CTLA-4 blockade augments systemic immune responses and has shown long-lasting effects in preclinical experiments as well as in clinical trials. OBJECTIVE: The study investigates the vaccination capacity of TCI in combination with the checkpoint inhibitor CTLA-4 in matters of primary response, memory formation and tumor protection and characterizes the role of regulatory T cells (Tregs). METHODS: After performing TCI with IMI-Sol (containing 5% Imiquimod) and the model epitope SIINFEKL, 6-8 week old C57BL/6 mice received anti-CTLA-4 antibody either s.c or i.p. The CTL responses and frequency of peptide specific CD8+ T-cells were then evaluated on day 8. To determine anti-tumor effects, a therapeutic tumor challenge with B16 OVA melanoma was performed. RESULTS: The combination of s.c. anti-CTLA-4 antibody and TCI leads to an enhanced systemic cytotoxic response, to memory formation and allows significantly improved survival in a tumor setting with B16 OVA melanoma. Towards the mechanism, we show that in this vaccination protocol the CTLA-4 antibody acts mainly Treg-independent. CONCLUSION: We demonstrate that the combination of TCI with IMI-Sol and anti-CTLA-4 can confer potent immune responses and tumor-protection. These results might contribute to the development of advanced vaccination approaches targeting tumors or persistent infectious diseases.

Transcutaneous immunization with a novel imiquimod nanoemulsion induces superior T cell responses and virus protection.

BACKGROUND: Transcutaneous immunization (TCI) is a novel vaccination strategy utilizing the skin associated lymphatic tissue to induce immune responses. TCI using a cytotoxic T lymphocyte (CTL) epitope and the Toll-like receptor 7 (TLR7) agonist imiquimod mounts strong CTL responses by activation and maturation of skin-derived dendritic cells (DCs) and their migration to lymph nodes. However, TCI based on the commercial formulation Aldara only induces transient CTL responses that needs further improvement for the induction of durable therapeutic immune responses. OBJECTIVE: Therefore we aimed to develop a novel imiquimod solid nanoemulsion (IMI-Sol) for TCI with superior vaccination properties suited to induce high quality T cell responses for enhanced protection against infections. METHODS: TCI was performed by applying a MHC class I or II restricted epitope along with IMI-Sol or Aldara (each containing 5% Imiquimod) on the shaved dorsum of C57BL/6, IL-1R, Myd88, Tlr7 or Ccr7 deficient mice. T cell responses as well as DC migration upon TCI were subsequently analyzed by flow cytometry. To determine in vivo efficacy of TCI induced immune responses, CTL responses and frequency of peptide specific T cells were evaluated on day 8 or 35 post vaccination and protection in a lymphocytic choriomeningitis virus (LCMV) infection model was assessed. RESULTS: TCI with the imiquimod formulation IMI-Sol displayed equal skin penetration of imiquimod compared to Aldara, but elicited superior CD8+ as well as CD4+ T cell responses. The induction of T-cell responses induced by IMI-Sol TCI was dependent on the TLR7/MyD88 pathway and independent of IL-1R. IMI-Sol TCI activated skin-derived DCs in skin-draining lymph nodes more efficiently compared to Aldara leading to enhanced protection in a LCMV infection model. CONCLUSION: Our data demonstrate that IMI-Sol TCI can overcome current limitations of previous imiquimod based TCI approaches opening new perspectives for transcutaneous vaccination strategies and allowing the use of this enhanced cutaneous drug-delivery system to be tailored for the improved prevention and treatment of infectious diseases and cancers.

Phenotypic and functional characterization of neutrophils and monocytes from patients with myelodysplastic syndrome by flow cytometry.

Myelodysplastic syndrome (MDS) is a clonal stem cell disorder frequently associated with inefficient granulopoiesis showing dysplastic polymorphonuclear neutrophils (PMNs). To assess PMN functionality in MDS in a clinical routine setting, 30 MDS patients and ten healthy volunteers were analyzed for PMN and monocyte phenotype and function (degranulation, CD62L shedding, oxidative burst and phagocytosis) upon stimulation with lipopolysaccharide by multi-color flow cytometry (MCFC). Our data show a heterogeneous pattern for CD66, CD16 and CD64 expression on PMNs of MDS patients. CD62L shedding rate and CD66 degranulation were reduced. Interestingly, we detected correlations between the WHO adapted prognostic scoring system (WPSS) and CD16 expression on PMNs as well as the international prognostic scoring system (IPSS) and CD11b degranulation by MCFC, suggesting clinical relevance of MCFC based function testing. In conclusion, MCFC of myelodysplastic immunophenotypes and PMN functionality are applicable in clinical settings, but further prospective studies are needed to assess the practical clinical value of such analyses.

Relationship of cortisol levels and genetic polymorphisms to antidepressant response to placebo and fluoxetine in patients with major depressive disorder: a prospective study.

BACKGROUND: Increased cortisol levels and genetic polymorphisms have been related to both major depressive disorder and antidepressant treatment outcome. The aim of this study is to evaluate the relationship between circadian salivary cortisol levels, cortisol suppression by dexamethasone and genetic polymorphisms in some HPA axis-related genes to the response to placebo and fluoxetine in depressed patients. METHODS: The diagnosis and severity of depression were performed using the Mini International Neuropsychiatric Interview (M.I.N.I.) and Hamilton depression scale (HAM-D17), respectively. Euthyroid patients were treated with placebo (one week) followed by fluoxetine (20 mg) (two months). Severity of depression was re-evaluated after placebo, three weeks and two months of fluoxetine treatments. Placebo response was defined as HAM-D17 score reductions of at least 25% and to < 15. Early response and response were reductions of at least 50% after three weeks and two months, and remission with ≤ 7 after two months. Plasma TSH, free-T4, circadian salivary cortisol levels and cortisol suppression by dexamethasone were evaluated. Seven genetic polymorphisms located in the Corticotrophin-releasing-hormone-receptor-1 (rs242939, rs242941, rs1876828), Corticotrophin-releasing-hormone-receptor-2 (rs2270007), Glucocorticoid-receptor (rs41423247), FK506-binding-protein-5 (rs1360780), and Arginine-vasopressin (rs3729965) genes were determined. Association analyses between response to placebo/fluoxetine and polymorphism were performed by chi-square or Fisher exact test. Cortisol levels were compared by t-test, ANOVA and the general linear model for repeated measures. RESULTS: 208 depressed patients were recruited, 187 of whom were euthyroid. Placebo responders, fluoxetine responders and remitters exhibited significantly lower circadian cortisol levels than those who did not respond (p-values of 0.014, 0.008 and 0.021 respectively). Patients who abandoned treatment before the third week also exhibited a trend to low cortisol levels (p = 0.057). The polymorphisms rs242939 (CRHR1) and rs2270007 (CRHR2) were not in Hardy-Weinberg equilibrium. Only the rs242939 polymorphism (CRHR1) exhibited association with early response (three weeks) to fluoxetine (p-value = 0.043). No other association between outcomes and polymorphisms was observed. CONCLUSIONS: These results support the clinical relevance of low salivary cortisol levels as a predictor of antidepressant response, either to placebo or to fluoxetine. Only one polymorphism in the CRHR1 gene was associated with the early response. Other factors may be involved in antidepressant response, although further studies are needed to identify them.

c.194 A>C (Q65P) mutation in the LMX1B gene in patients with nail-patella syndrome associated with glaucoma.

PURPOSE: To report the clinical, ophthalmic, extraophthalmic, and genetic characteristics of nail-patella syndrome (NPS) in a Chilean family and to investigate the expressivity of open angle glaucoma (OAG) and ocular hypertension (OHT) in the family members. METHODS: Five family members affected with NPS and two unaffected members underwent a complete ophthalmologic examination, including computerized visual field, optical coherence tomography (OCT) of the optic disc and ultrasound pachymetry. Renal function was assessed by urinalysis and blood tests. Orthopedic evaluations were also performed, including radiological studies of the wrist, elbow and hip joints. Genomic DNA was extracted from peripheral leukocytes of the five affected and two unaffected family members. Exons 2-6 of the LIM homeobox transcription factor 1-beta (LMX1B) gene were screened for mutations by DNA sequencing of the proband. We also screened for mutations in exon 2 by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the other participants and 91 blood donors. RESULTS: Five living family members from three generations were positively diagnosed with NPS, three of them with varying degrees of OAG and one with OHT. Retinal nerve fiber layer (RNFL) thickness measured by spectral domain OCT was below normal values in three individuals. All subjects evaluated had normal nephrologic function. Orthopedic, clinical, and radiological alterations were compatible with NPS. Screening for mutations in exons 2- 6 of LMX1B showed a heterozygous missense mutation c.194 A>C changing glutamine to proline within exon 2 in codon 65 (Q65P) of the coding sequence. This mutation was present in all NPS subjects and absent in the unaffected family members and in 91 Chilean blood donors. CONCLUSIONS: This is the first report of c.194 A>C mutation in LMX1B in a Chilean family with NPS and the second worldwide. The phenotype associated with this mutation is variable within the family, although we noted a close connection between the presence of the c.194 A>C mutation and the presence of OHT or OAG and probably also with an early onset of OHT in patients with NPS. All subjects older than 21 years had either OHT or OAG. We also suggest that the LMX1B mutation may be related to affective disorders.