Exploring atypical manifestations and multisystem involvement of Epstein-Barr virus infection in hospitalized pediatric patients from Mexico: insights from a tertiary hospital (2012-2022).

INTRODUCTION: Epstein-Barr virus (EBV) infection, with a global prevalence exceeding 95%, typically manifests in children as infectious mononucleosis. However, clinical practice frequently encounters diverse atypical presentations characterized by multisystem involvement, often resulting in an unfavorable clinical course. Our objective is to describe the clinical manifestations and results of EBV infection in a tertiary pediatric hospital in Mexico. METHOD: An observational, transversal, retrospective, and descriptive study that included a systematic review of medical records (2012-2022) of patients under 18 years of age with detectable EBV particles in peripheral blood. RESULTS: The study included 26 patients with a median age of 5 years and a male predominance of 53.8%. Predominant symptoms were fever (85%) and lymphadenopathy (35%). Sixty-five percent had severe and atypical manifestations, including pneumonia and hepatic, hematologic-oncologic, and autoimmune diseases. Anemia, thrombocytopenia and leukopenia were common, with lymphocytosis in 19% of cases. The median EBV viral load was 2816 copies/mL (range: 555-355,500 copies/mL). Four deaths related to EBV infection were reported. Viral load in these cases also varied widely from 594 to 121,000 copies/mL. Supportive care was administered to 85% of patients, while others received antiviral treatment, steroids, and rituximab. CONCLUSION: Atypical manifestations were common, especially in children with multisystem involvement. EBV should be considered as a potential contributor to a diverse spectrum of clinical presentations, emphasizing the need for comprehensive evaluation and awareness in clinical diagnosis.

INTRODUCCIÓN: La infección por el virus de Epstein-Barr (VEB) tiene una prevalencia mundial superior al 95%. Se considera que en los niños se manifiesta principalmente como mononucleosis infecciosa; sin embargo, en la práctica clínica, a menudo encontramos numerosas manifestaciones atípicas con compromiso multisistémico que llevan a un curso desfavorable. Nuestro objetivo es describir las manifestaciones clínicas y los resultados de la infección por VEB en un hospital pediátrico de tercer nivel en México. MÉTODO: Estudio observacional, transversal, retrospectivo y descriptivo, en el cual se revisaron sistemáticamente los expedientes médicos de pacientes menores de 18 años con una detección positiva de partículas de VEB en sangre periférica en el periodo 2012-2022. RESULTADOS: Se incluyeron 26 pacientes con una mediana de edad de 5 años y predominio de varones (53.8%). El 65% presentaron manifestaciones graves y atípicas, incluyendo enfermedades respiratorias, hepáticas, hematooncológicas y autoinmunitarias. Los síntomas más frecuentes fueron fiebre (85%) y linfadenopatía (35%). El 54% presentaron manifestaciones atípicas, incluyendo linfohistiocitosis hemofagocítica, neumonía y neoplasia. La anemia, la trombocitopenia y la leucocitopenia fueron comunes, mientras que el 19% presentaron linfocitosis. La media de la carga viral fue de 2816 copias/ml (555-355,500). Se informaron cuatro muertes atribuidas a la infección por VEB, con valores de carga viral de 594 a 121,000 copias/ml. El 85% de los pacientes recibieron solo tratamiento sintomático, mientras que otros recibieron antivirales, esteroides y rituximab. CONCLUSIÓN: Las manifestaciones atípicas se observaron comúnmente, en especial en niños con compromiso multisistémico. El VEB debe considerarse como un potencial factor contribuyente en el diagnóstico de una amplia gama de manifestaciones clínicas.

Impact on Fatality Rates and Years of Life Lost During the COVID-19 Pandemic: The Experience of the Mexican Public Health Incident Management Command.

BACKGROUND: The SARS-CoV-2 pandemic challenged health systems worldwide. In Mexico, the Public Health Incident Management Command (COISS) strategy was implemented to improve health care for patients with COVID-19 who required hospitalization. AIM: To evaluate the impact of the COISS strategy on case fatality rates (CFR) and years of life lost (YLL) in hospitalized patients with COVID-19. MATERIALS AND METHODS: The COISS strategy included eight actions implemented in states with high epidemic risk (COISS states). A secondary analysis of the public database from the Mexican Ministry of Health was performed considering patients with confirmed diagnoses of SARS-CoV-2 infection. The COISS strategy effectiveness was evaluated by its impact on in-hospital CFR and YLL at the beginning (T0) and end (T1) of the third wave, and at the end of the fourth wave (T2) and compared to states without intervention (non-COISS states). RESULTS: At T0, COISS states showed a higher CFR for hospitalized patients than non-COISS states, which decreased after the strategy implementation. After correction for baseline conditions, lower relative CFR at T1 and T2, compared to T0, and a protective effect in different age groups, especially in those ≥65 years, were found in hospitalized patients in COISS states. The COISS strategy was associated with lower CFR in hospitalized patients with COVID-19 at both T1 and T2. At T0, YLLs were higher in COISS states, but there were no significant differences at T1 and T2. CONCLUSIONS: COISS interventions effectively reduced CFR in hospitalized patients with COVID-19, providing protection to vulnerable patients and reducing the YLL gap.

TCF1-positive and TCF1-negative TRM CD8 T cell subsets and cDC1s orchestrate melanoma protection and immunotherapy response.

BACKGROUND: Melanoma, the most lethal form of skin cancer, has undergone a transformative treatment shift with the advent of checkpoint blockade immunotherapy (CBI). Understanding the intricate network of immune cells infiltrating the tumor and orchestrating the control of melanoma cells and the response to CBI is currently of utmost importance. There is evidence underscoring the significance of tissue-resident memory (TRM) CD8 T cells and classic dendritic cell type 1 (cDC1) in cancer protection. Transcriptomic studies also support the existence of a TCF7+ (encoding TCF1) T cell as the most important for immunotherapy response, although uncertainty exists about whether there is a TCF1+TRM T cell due to evidence indicating TCF1 downregulation for tissue residency activation. METHODS: We used multiplexed immunofluorescence and spectral flow cytometry to evaluate TRM CD8 T cells and cDC1 in two melanoma patient cohorts: one immunotherapy-naive and the other receiving immunotherapy. The first cohort was divided between patients free of disease or with metastasis 2 years postdiagnosis while the second between CBI responders and non-responders. RESULTS: Our study identifies two CD8+TRM subsets, TCF1+ and TCF1-, correlating with melanoma protection. TCF1+TRM cells show heightened expression of IFN-γ and Ki67 while TCF1- TRM cells exhibit increased expression of cytotoxic molecules. In metastatic patients, TRM subsets undergo a shift in marker expression, with the TCF1- subset displaying increased expression of exhaustion markers. We observed a close spatial correlation between cDC1s and TRMs, with TCF1+TRM/cDC1 pairs enriched in the stroma and TCF1- TRM/cDC1 pairs in tumor areas. Notably, these TCF1- TRMs express cytotoxic molecules and are associated with apoptotic melanoma cells. Both TCF1+ and TCF1- TRM subsets, alongside cDC1, prove relevant to CBI response. CONCLUSIONS: Our study supports the importance of TRM CD8 T cells and cDC1 in melanoma protection while also highlighting the existence of functionally distinctive TCF1+ and TCF1- TRM subsets, both crucial for melanoma control and CBI response.

Newcastle disease virus vector-based SARS-CoV-2 vaccine candidate AVX/COVID-12 activates T cells and is recognized by antibodies from COVID-19 patients and vaccinated individuals.

Introduction: Several effective vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been developed and implemented in the population. However, the current production capacity falls short of meeting global demand. Therefore, it is crucial to further develop novel vaccine platforms that can bridge the distribution gap. AVX/COVID-12 is a vector-based vaccine that utilizes the Newcastle Disease virus (NDV) to present the SARS-CoV-2 spike protein to the immune system. Methods: This study aims to analyze the antigenicity of the vaccine candidate by examining antibody binding and T-cell activation in individuals infected with SARS-CoV-2 or variants of concern (VOCs), as well as in healthy volunteers who received coronavirus disease 2019 (COVID-19) vaccinations. Results: Our findings indicate that the vaccine effectively binds antibodies and activates T-cells in individuals who received 2 or 3 doses of BNT162b2 or AZ/ChAdOx-1-S vaccines. Furthermore, the stimulation of T-cells from patients and vaccine recipients with AVX/COVID-12 resulted in their proliferation and secretion of interferon-gamma (IFN-γ) in both CD4+ and CD8+ T-cells. Discussion: The AVX/COVID-12 vectored vaccine candidate demonstrates the ability to stimulate robust cellular responses and is recognized by antibodies primed by the spike protein present in SARS-CoV-2 viruses that infected patients, as well as in the mRNA BNT162b2 and AZ/ChAdOx-1-S vaccines. These results support the inclusion of the AVX/COVID-12 vaccine as a booster in vaccination programs aimed at addressing COVID-19 caused by SARS-CoV-2 and its VOCs.

Strengthening Essential Health Services Recovery Policy after the COVID-19 Pandemic. Evidence from the Mexican Institute of Social Security.

AIM: To evaluate the progress of the Mexican Institute of Social Security Recovery Policy (IMSS-RP) in addressing the decline in essential health services caused by the COVID-19 pandemic. METHODS: We analyzed eleven indicators of essential health services from 35 IMSS state delegations. The assessment included ambulatory and hospital care indicators such as breast and cervical cancer screening, family medicine, dental and specialty visits, diabetes and hypertension visits and health outcomes, deliveries, and elective surgeries. We analyzed the period before (January 2018-March 2021) and during (April 2021-June 2023) the implementation of the IMSS-RP. Statistical analysis to determine the association of the policy with service indicators and the change in their trends included an interrupted time series analysis and Poisson Generalized Estimating Equation models. RESULTS: The volume of services showed substantial declines during the first year of the COVID-19 pandemic, reaching between 11 and 81% of pre-pandemic levels. All services increased significantly during the first 27 months of the IMSS-RP implementation; specialty visits, cervical and breast cancer screening, and diabetes control exceeded pre-pandemic levels (103%,112%,103%, and 138%, respectively). However, only deliveries and the percentage of patients with controlled diabetes and hypertension showed a stable increase following the IMSS-RP implementation, whereas the remaining services showed an initial increase but began to decrease over time. CONCLUSIONS: After 27 months of implementation, IMSS-RP achieved progress in increasing the volume of essential health services and improving chronic disease control. However, declining trends in several services signal the need to focalize the policy.

The Immune Microenvironment Landscape of Pituitary NeuroEndocrine Tumors, a Transcriptomic Approach.

Pituitary neuroendocrine tumors (PitNET) are known to be variably infiltrated by different immune cells. Nonetheless, their role in pituitary oncogenesis has only begun to be unveiled. The immune microenvironment could determine the biological and clinical behavior of a neoplasm and may have prognostic implications. To evaluate the expression of immune-related genes and to correlate such expression with the presence of infiltrating immune cells in forty-two PitNETs of different lineages, we performed whole transcriptome analysis and RT-qPCR. Deconvolution analysis was carried out to infer the immune cell types present in each tumor and the presence of immune cells was confirmed by immunofluorescence. We found characteristic expression profiles of immune-related genes including those encoding interleukins and chemokines for each tumor lineage. Genes such as IL4-I1, IL-36A, TIRAP, IL-17REL, and CCL5 were upregulated in all PitNETS, whereas IL34, IL20RA, and IL-2RB characterize the NR5A1-, TBX19-, and POU1F1-derived tumors, respectively. Transcriptome deconvolution analysis showed that M2 macrophages, CD4+ T cells, CD8+ T cells, NK cells, and neutrophils can potentially infiltrate PitNET. Furthermore, CD4+ and CD8+ T cells and NK cells infiltration was validated by immunofluorescence. Expression of CCL18, IL-5RA, and HLA-B as well as macrophage tumor infiltration could identify patients who can potentially benefit from treatment with immune checkpoint inhibitors.

The cholera toxin B subunit induces trained immunity in dendritic cells and promotes CD8 T cell antitumor immunity.

Introduction: Innate immune training is a metabolic, functional, and epigenetic long-term reprogramming of innate cells triggered by different stimuli. This imprinting also reaches hematopoietic precursors in the bone marrow to sustain a memory-like phenotype. Dendritic cells (DCs) can exhibit memory-like responses, enhanced upon subsequent exposure to a pathogen; however, whether this imprinting is lineage and stimulus-restricted is still being determined. Nevertheless, the functional consequences of DCs training on the adaptive and protective immune response against non-infectious diseases remain unresolved. Methods: We evaluated the effect of the nontoxic cholera B subunit (CTB), LPS and LTA in the induction of trained immunity in murine DCs revealed by TNFa and LDH expression, through confocal microscopy. Additionally, we obtained bone marrow DCs (BMDCs) from mice treated with CTB, LPS, and LTA and evaluated training features in DCs and their antigen-presenting cell capability using multiparametric cytometry. Finally, we design an experimental melanoma mouse model to demonstrate protection induced by CTB-trained DCs in vivo. Results: CTB-trained DCs exhibit increased expression of TNFa, and metabolic reprogramming indicated by LDH expression. Moreover, CTB training has an imprint on DC precursors, increasing the number and antigen-presenting function in BMDCs. We found that training by CTB stimulates the recruitment of DC precursors and DCs infiltration at the skin and lymph nodes. Interestingly, training-induced by CTB promotes a highly co-stimulatory phenotype in tumor-infiltrating DCs (CD86+) and a heightened functionality of exhausted CD8 T cells (Ki67+, GZMB+), which were associated with a protective response against melanoma challenge in vivo. Conclusion: Our work indicates that CTB can induce innate immune training on DCs, which turns into an efficient adaptive immune response in the melanoma model and might be a potential immunotherapeutic approach for tumor growth control.

Outbreak of Fusarium solani Meningitis in Immunocompetent Persons Associated With Neuraxial Blockade in Durango, Mexico, 2022-2023.

Background: Fungal meningitis can be associated with epidural anesthesia procedures. Fusariosis is a rare infection typically affecting immunocompromised patients and rarely causes meningitis. During 2022-2023, public health officials responded to a large outbreak of Fusarium solani meningitis associated with epidural anesthesia in Durango, Mexico. Methods: The public health response and epidemiological and clinical features of patients affected by this outbreak were described. Coordinated actions were addressed to identify the etiological agent, determine its drug susceptibility, develop diagnostic tests, and implement clinical and epidemiological protocols. Retrospective analyses of clinical variables and outcomes were performed to determine association with better patient survival. Results: A total of 1801 persons exposed to epidural anesthesia were identified, of whom 80 developed meningitis. Fusarium solani was found in 3 brain biopsies and showed susceptibility to voriconazole and amphotericin B. After F solani polymerase chain reaction (PCR) implementation, 57 patients with meningitis were PCR-screened, and 31 (38.8%) had a positive result. Most patients were female (95%), and cesarean section was the most common surgical procedure (76.3%). The case fatality rate was 51.3% (41 patients) and the median hospitalization duration was 39.5 days (interquartile range, 18-86 days). Seventy-one patients (88.8%) received voriconazole/amphotericin B and 64 subjects (80%) additionally received steroids. Cox regression analysis showed an increased lethality risk in patients who received antifungal treatment after 5 days (hazard ratio, 2.1 [95% confidence interval, 1.01-4.48], P < .05). Conclusions: The F solani meningitis outbreak in Durango was an unprecedented medical challenge. Timely treatment and effective healthcare management were associated with better survival outcomes.

The invasive margin of early-stage human colon tumors is infiltrated with neutrophils of an antitumoral phenotype.

Neutrophils infiltrate several types of cancer; however, whether their presence is associated with disease progression remains controversial. Here, we show that colon tumors overexpress neutrophil chemoattractants compared to healthy tissues, leading to their recruitment to the invasive margin and the central part of colon tumors. Of note, tumor-associated neutrophils expressing tumor necrosis factor α, which usually represents an antitumoral phenotype, were predominantly located in the invasive margin. Tumor-associated neutrophils from the invasive margin displayed an antitumoral phenotype with higher ICAM-1 and CD95 expression than neutrophils from healthy adjacent tissues. A higher neutrophil/lymphocyte ratio was found at later stages compared to the early phases of colon cancer. A neutrophil/lymphocyte ratio ≤3.5 predicted tumor samples had significantly more neutrophils at the invasive margin and the central part. Moreover, tumor-associated neutrophils at the invasive margin of early-stage tumors showed higher ICAM-1 and CD95 expression. Coculture of colon cancer cell lines with primary neutrophils induced ICAM-1 and CD95 expression, confirming our in situ findings. Thus, our data demonstrate that tumor-associated neutrophils with an antitumoral phenotype characterized by high ICAM-1 and CD95 expression infiltrate the invasive margin of early-stage colon tumors, suggesting that these cells can combat the disease at its early courses. The presence of tumor-associated neutrophils with antitumoral phenotype could help predict outcomes of patients with colon cancer.

Potential biomarkers for fatal outcome prognosis in a cohort of hospitalized COVID-19 patients with pre-existing comorbidities.

The difficulty in predicting fatal outcomes in patients with coronavirus disease 2019 (COVID-19) impacts the general morbidity and mortality due to severe acute respiratory syndrome-coronavirus 2 infection, as it wears out the hospital services that care for these patients. Unfortunately, in several of the candidates for prognostic biomarkers proposed, the predictive power is compromised when patients have pre-existing comorbidities. A cohort of 147 patients hospitalized for severe COVID-19 was included in a descriptive, observational, single-center, and prospective study. Patients were recruited during the first COVID-19 pandemic wave (April-November 2020). Data were collected from the clinical history whereas immunophenotyping by multiparameter flow cytometry analysis allowed us to assess the expression of surface markers on peripheral leucocyte. Patients were grouped according to the outcome in survivors or non-survivors. The prognostic value of leucocyte, cytokines or HLA-DR, CD39, and CD73 was calculated. Hypertension and chronic renal failure but not obesity and diabetes were conditions more frequent among the deceased patient group. Mixed hypercytokinemia, including inflammatory (IL-6) and anti-inflammatory (IL-10) cytokines, was more evident in deceased patients. In the deceased patient group, lymphopenia with a higher neutrophil-lymphocyte ratio (NLR) value was present. HLA-DR expression and the percentage of CD39+ cells were higher than non-COVID-19 patients but remained similar despite the outcome. Receiver operating characteristic analysis and cutoff value of NLR (69.6%, 9.4), percentage NLR (pNLR; 71.1%, 13.6), and IL-6 (79.7%, 135.2 pg/mL). The expression of HLA-DR, CD39, and CD73, as many serum cytokines (other than IL-6) and chemokines levels do not show prognostic potential, were compared to NLR and pNLR values.

CXCR5 and TIM-3 expressions define distinct exhausted T cell subsets in experimental cutaneous infection with Leishmania mexicana.

T-cell exhaustion is a key stage in chronic infections since it limits immunopathology, but also hinders the elimination of pathogens. Exhausted T (Tex) cells encompass dynamic subsets, including progenitor cells that sustain long-term immunity through their memory/stem like properties, and terminally-differentiated cells, resembling the so-called Tex cells. The presence of Tex cells in chronic leishmaniasis has been reported in humans and murine models, yet their heterogeneity remains unexplored. Using flow cytometry, we identified Tex cells subtypes based on PD-1, CXCR5 and TIM-3 expressions in draining lymph nodes (dLNs) and lesion sites of C57BL/6 mice infected with L. mexicana at 30-, 60- and 90-days post-infection. We showed that infected mice developed a chronic infection characterized by non-healing lesions with a high parasite load and impaired Th1/Th2 cytokine production. Throughout the infection, PD-1+ cells were observed in dLNs, in addition to an enhanced expression of PD-1 in both CD4+ and CD8+ T lymphocytes. We demonstrated that CD4+ and CD8+ T cells were subdivided into PD-1+CXCR5+TIM-3- (CXCR5+), PD-1+CXCR5+TIM-3+ (CXCR5+TIM-3+), and PD-1+CXCR5-TIM-3+ (TIM-3+) subsets. CXCR5+ Tex cells were detected in dLNs during the whole course of the infection, whereas TIM-3+ cells were predominantly localized in the infection sites at day 90. CXCR5+TIM-3+ cells only increased at 30 and 60 days of infection in dLNs, whereas no increase was observed in the lesions. Phenotypic analysis revealed that CXCR5+ cells expressed significantly higher levels of CCR7 and lower levels of CX3CR1, PD-1, TIM-3, and CD39 compared to the TIM-3+ subset. CXCR5+TIM-3+ cells expressed the highest levels of all exhaustion-associated markers and of CX3CR1. In agreement with a less exhausted phenotype, the frequency of proliferating Ki-67 and IFN-γ expressing cells was significantly higher in the CXCR5+ subset within both CD4+ and CD8+ T cells compared to their respective TIM-3+ subsets, whereas CD8+CXCR5+TIM-3+ and CD8+TIM-3+ subsets showed an enhanced frequency of degranulating CD107a+ cells. In summary, we identified a novel, less-differentiated CXCR5+ Tex subset in experimental cutaneous leishmaniasis caused by L. mexicana. Targeting these cells through immune checkpoint inhibitors such as anti-PD-1 or anti PD-L1 might improve the current treatment for patients with the chronic forms of leishmaniasis.

Acral Melanoma Is Infiltrated with cDC1s and Functional Exhausted CD8 T Cells Similar to the Cutaneous Melanoma of Sun-Exposed Skin.

Acral melanoma (AM) is the most common melanoma in non-Caucasian populations, yet it remains largely understudied. As AM lacks the UV-radiation mutational signatures that characterize other cutaneous melanomas, it is considered devoid of immunogenicity and is rarely included in clinical trials assessing novel immunotherapeutic regimes aiming to recover the antitumor function of immune cells. We studied a Mexican cohort of melanoma patients from the Mexican Institute of Social Security (IMSS) (n = 38) and found an overrepresentation of AM (73.9%). We developed a multiparametric immunofluorescence technique coupled with a machine learning image analysis to evaluate the presence of conventional type 1 dendritic cells (cDC1) and CD8 T cells in the stroma of melanoma, two of the most relevant immune cell types for antitumor responses. We observed that both cell types infiltrate AM at similar and even higher levels than other cutaneous melanomas. Both melanoma types harbored programmed cell death protein 1 (PD-1+) CD8 T cells and PD-1 ligand (PD-L1+) cDC1s. Despite this, CD8 T cells appeared to preserve their effector function and expanding capacity as they expressed interferon-γ (IFN-γ) and KI-67. The density of cDC1s and CD8 T cells significantly decreased in advanced stage III and IV melanomas, supporting these cells' capacity to control tumor progression. These data also argue that AM could respond to anti-PD-1-PD-L1 immunotherapy.

Subclassification of B-acute lymphoblastic leukemia according to age, immunophenotype and microenvironment, predicts MRD risk in Mexican children from vulnerable regions.

Introduction: The decisive key to disease-free survival in B-cell precursor acute lymphoblastic leukemia in children, is the combination of diagnostic timeliness and treatment efficacy, guided by accurate patient risk stratification. Implementation of standardized and high-precision diagnostic/prognostic systems is particularly important in the most marginalized geographic areas in Mexico, where high numbers of the pediatric population resides and the highest relapse and early death rates due to acute leukemias are recorded even in those cases diagnosed as standard risk. Methods: By using a multidimensional and integrated analysis of the immunophenotype of leukemic cells, the immunological context and the tumor microenvironment, this study aim to capture the snapshot of acute leukemia at disease debut of a cohort of Mexican children from vulnerable regions in Puebla, Oaxaca and Tlaxcala and its potential use in risk stratification. Results and discussion: Our findings highlight the existence of a distinct profile of ProB-ALL in children older than 10 years, which is associated with a six-fold increase in the risk of developing measurable residual disease (MRD). Along with the absence of CD34+ seminal cells for normal hematopoiesis, this ProB-ALL subtype exhibited several characteristics related to poor prognosis, including the high expression level of myeloid lineage markers such as MPO and CD33, as well as upregulation of CD19, CD34, CD24, CD20 and nuTdT. In contrast, it showed a trend towards decreased expression of CD9, CD81, CD123, CD13, CD15 and CD21. Of note, the mesenchymal stromal cell compartment constituting their leukemic niche in the bone marrow, displayed characteristics of potential suppressive microenvironment, such as the expression of Gal9 and IDO1, and the absence of the chemokine CXCL11. Accordingly, adaptive immunity components were poorly represented. Taken together, our results suggest, for the first time, that a biologically distinct subtype of ProB-ALL emerges in vulnerable adolescents, with a high risk of developing MRD. Rigorous research on potential enhancing factors, environmental or lifestyle, is crucial for its detection and prevention. The use of the reported profile for early risk stratification is suggested.

Cancer Incidence in Merida, Mexico 2015-2018: First Report from the Population-based Cancer Registry.

BACKGROUND: Cancer registries are essential for monitoring cancer burden and patterns, and document changes in time for cancer control. Hereby, we present the first results of four years of the Merida population-based cancer registry in Mexico. METHODS: The registry collects data on all new cancers diagnosed since 2015 using both active and passive methods including a total of 104 information sources. Definitions and coding follow international standards. Using CanReg5 software, age-standardized incidence rates (ASR/100,000 person years) were computed by direct method using the world standard population. RESULTS: A total of 5684 new cancer cases were registered during 2015-2018, 2321 in males and 3363 in females corresponding to age-adjusted incidence rates (ASR per 100,000) of 128.5, and 153.1, respectively. Most frequent cancers among males were prostate cancer (ASR 29.8), lymphomas (ASR 10.9) and colorectal cancer (ASR 9.7) while among females it was breast cancer (ASR 49.3), cervical cancer (ASR 17.5) and corpus uteri (ASR 11.5). Childhood cancers (0-14 year) represented 2.9% of all cancers, with leukemias accounting for 52% of the new cases. Overall, 87.6% of new cases were microscopically verified. CONCLUSIONS: The data reported provide information on the cancer profile in Merida. Prostate and breast cancer are the main incident cancers. Cervical cancers present high rates among women, while lymphomas and liver cancer data merit further exploration. Efforts to support the Merida cancer registry as well as other registries in Mexico need to be pursued in order to have locally recorded data to support cancer control measures.

A Machine Learning Workflow of Multiplexed Immunofluorescence Images to Interrogate Activator and Tolerogenic Profiles of Conventional Type 1 Dendritic Cells Infiltrating Melanomas of Disease-Free and Metastatic Patients.

Melanoma is the deadliest form of skin cancer. Due to its high mutation rates, melanoma is a convenient model to study antitumor immune responses. Dendritic cells (DCs) play a key role in activating cytotoxic CD8+ T lymphocytes and directing them to kill tumor cells. Although there is evidence that DCs infiltrate melanomas, information about the profile of these cells, their activity states, and potential antitumor function remains unclear, particularly for conventional DCs type 1 (cDC1). Approaches to profiling tumor-infiltrating DCs are hindered by their diversity and the high number of signals that can affect their state of activation. Multiplexed immunofluorescence (mIF) allows the simultaneous analysis of multiple markers, but image-based analysis is time-consuming and often inconsistent among analysts. In this work, we evaluated several machine learning (ML) algorithms and established a workflow of nine-parameter image analysis that allowed us to study cDC1s in a reproducible and accessible manner. Using this workflow, we compared melanoma samples between disease-free and metastatic patients at diagnosis. We observed that cDC1s are more abundant in the tumor infiltrate of the former. Furthermore, cDC1s in disease-free patients exhibit an expression profile more congruent with an activator function: CD40highPD-L1low CD86+IL-12+. Although disease-free patients were also enriched with CD40-PD-L1+ cDC1s, these cells were also more compatible with an activator phenotype. The opposite was true for metastatic patients at diagnosis who were enriched for cDC1s with a more tolerogenic phenotype (CD40lowPD-L1highCD86-IL-12-IDO+). ML-based workflows like the one developed here can be used to analyze complex phenotypes of other immune cells and can be brought to laboratories with standard expertise and computer capacity.

The Protective Role of pVHL in Imiquimod-Induced Psoriasis-like Skin Inflammation.

Psoriasis is a chronic inflammatory disease distinguished by an excessive proliferation and abnormal differentiation of keratinocytes. Immune cells, such as T lymphocytes and neutrophils, and inflammatory cytokines, such as Tumor Necrosis Factor-α (TNF-α) and interleukin 17 (IL-17), are essential for maintaining psoriatic lesions. Additionally, a hypoxic milieu present in the skin promotes the expression of transcriptional factor hypoxia-inducible factor-1 alpha (HIF-1α). This protein regulates the expression of angiogenic and glycolytic factors, such as vascular endothelial grown factor and lactate dehydrogenase (LDH), both relevant in chronic inflammation. The von Hippel-Lindau protein (pVHL) is a negative regulator of HIF-1α. Previously, we found that pVHL was almost absent in the lesions of psoriasis patients; therefore, we investigated the impact of rescue pVHL expression in lesional skin. We used the imiquimod-induced psoriasis-like mouse model as an adenoviral vector that allowed us to express pVHL in the skin. Our data show that, in lesional skin, pVHL expression was reduced, whereas HIF-1α was increased. Remarkably, the retrieval of pVHL prevented psoriatic lesions, diminishing erythema, scale, and epidermal and vascular thickness. Furthermore, pVHL expression was capable of reducing HIF-1α, LDH, TNF-α and immune cell infiltration (mainly IL-17+ neutrophils). In conclusion, our results demonstrate that pVHL has a protective role to play in the pathophysiology of psoriasis.

Antigen Targeting of Porcine Skin DEC205+ Dendritic Cells.

Dendritic cell (DC) targeting by DEC205+ cells effectively promotes the internalization of antigens that may trigger a specific immune response. In this study, we evaluated the ability of a recombinant antibody, anti-DEC205 (rAb ZH9F7), to trigger cellular endocytosis in subpopulations of DCs and targeted cells after intradermal injection and subsequent migration toward lymph nodes. Furthermore, the cellular immune response was evaluated in pigs after intradermal application of the antigenized rAb ZH9F7 combined with porcine circovirus type 2 cap antigen (rAb ZH9F7-Cap). We demonstrated that rAb ZH9F7 recognized conventional type 1 and 2 DCs from the blood and skin and monocytes. It promoted receptor-mediated endocytosis and migration of cDCs and moDCs toward regional lymph nodes. Intradermal application of rAb ZH9F7-Cap induced a higher frequency of IFN-γ-secreting CD4+CD8+ T lymphocytes and antibodies against Cap protein than that in the control group. In conclusion, the rAb ZH9F7-Cap system promoted the target of skin cDC1 and cDC2, provoking migration to the regional lymph nodes and inducing a Th1 response, as evidenced by the proliferation of double-positive CD4+CD8+ T cells, which correlates with an enhanced ability to target the cDC1 subset both in vitro and in vivo.

NLRP3 Regulates IL-4 Expression in TOX+ CD4+ T Cells of Cutaneous T Cell Lymphoma to Potentially Promote Disease Progression.

In cutaneous T cell lymphoma (CTCL), a dominant Th2 profile associated with disease progression has been proposed. Moreover, although the production and regulation of IL-4 expression during the early stages of the disease may have important implications in later stages, these processes are poorly understood. Here, we demonstrate the presence of TOX+ CD4+ T cells that produce IL-4+ in early-stage skin lesions of CTCL patients and reveal a complex mechanism by which the NLRP3 receptor promotes a Th2 response by controlling IL-4 production. Unassembled NLRP3 is able to translocate to the nucleus of malignant CD4+ T cells, where it binds to the human il-4 promoter. Accordingly, IL-4 expression is decreased by knocking down and increased by promoting the nuclear localization of NLRP3. We describe a positive feedback loop in which IL-4 inhibits NLRP3 inflammasome assembly, thereby further increasing its production. IL-4 induced a potentially malignant phenotype measured based on TOX expression and proliferation. This mechanism of IL-4 regulation mediated by NLRP3 is amplified in late-stage CTCL associated with disease progression. These results indicate that NLRP3 might be a key regulator of IL-4 expression in TOX+ CD4+ T cells of CTCL patients and that this mechanism might have important implications in the progression of the disease.

CD4+ and CD8+ Circulating Memory T Cells Are Crucial in the Protection Induced by Vaccination with Salmonella Typhi Porins.

Salmonella enterica serovar Typhi (S. Typhi) porins, OmpC and OmpF, are potent inducers of the immune response against S. Typhi in mice and humans. Vaccination with porins induces the protection against 500 LD50 of S. Typhi, life-lasting bactericidal antibodies and effector T cell responses in mice; however, the nature of the memory T cell compartment and its contribution to protection remains unknown. In this work, we firstly observed that vaccination with porins induces in situ (skin) CD4+ and CD8+ T cell responses. Analysis of the porin-specific functional responses of skin CD4+ and CD8+ T cells showed IFN-gamma- and IL-17-producing cells in both T cell populations. The memory phenotype of porin-specific T cells indicated the presence of resident and effector memory phenotypes in the skin, and a central memory phenotype in the skin-draining lymph node. In addition, we demonstrated that vaccination with porins via skin reduces the bacterial burden following challenge. Finally, evaluating the role of the circulating T cell memory population in protection, we showed that circulating memory CD4+ and CD8+ T cells are crucial in porin-mediated protection against S. Typhi. Overall, this study highlights the importance of inducing circulating memory T cell responses in order to achieve the optimal protection provided by porins, showing a mechanism that could be sought in the rational development of vaccines.

Induction of Therapeutic Protection in an HPV16-Associated Mouse Tumor Model Through Targeting the Human Papillomavirus-16 E5 Protein to Dendritic Cells.

HPV E5 is an oncoprotein mainly expressed in premalignant lesions, which makes it an important target for a vaccine to prevent or cure cervical cancer (CC). In this study, we evaluated whether E5 targeted to DEC-205, present in dendritic cells (DCs), could induce a therapeutic protection against HPV16-induced tumor cells in a mouse model. The HPV-16 E5 (16E5) protein was cross-linked to a monoclonal antibody (mAb) specific to mouse DEC-205 (anti-DEC-205:16E5) or to an isotype control mAb (isotype:16E5). Rotavirus VP6 was cross-linked to the mouse anti-DEC-205 mAb (anti-DEC-205:VP6) as a non-specific antigen control. BALB/c mice were inoculated subcutaneously (s.c.) with the 16E5-expressing BMK-16/myc tumor cells, and 7 and 14 days later the mice were immunized s.c. with the conjugates, free 16E5 or PBS in the presence of adjuvant. Tumor growth was monitored to evaluate protection. A strong protective immune response against the tumor cells was induced when the mice were inoculated with the anti-DEC-205:16E5 conjugate, since 70% of the mice controlled the tumor growth and survived, whereas the remaining 30% developed tumors and died by day 72. In contrast, 100% of the mice in the control groups died by day 30. The anti-DEC-205:16E5 conjugate was found to induce 16E5-specific memory T cells, with a Th1/Th17 profile. Both CD4+ and CD8+ T cells contributed to the observed protection. Finally, treating mice that had developed tumors with an anti-PD-1 mAb, delayed the tumor growth for more than 20 days. These results show that targeting 16E5 to DEC-205, alone or combined with an immune checkpoint blockade, could be a promising protocol for the treatment of the early stages of HPV-associated cancer.