Anti-tumor activity of a T-helper 1 multiantigen vaccine in a murine model of prostate cancer.

Prostate cancer is one of the few malignancies that includes vaccination as a treatment modality. Elements of an effective cancer vaccine should include the ability to elicit a Type I T-cell response and target multiple antigenic proteins expressed early in the disease. Using existing gene datasets encompassing normal prostate tissue and tumors with Gleason Score ≤ 6 and ≥ 8, 10 genes were identified that were upregulated and conserved in prostate cancer regardless of the aggressiveness of disease. These genes encoded proteins also expressed in prostatic intraepithelial neoplasia. Putative Class II epitopes derived from these proteins were predicted by a combination of algorithms and, using human peripheral blood, epitopes which selectively elicited IFN-γ or IL-10 dominant antigen specific cytokine secretion were determined. Th1 selective epitopes were identified for eight antigens. Epitopes from three antigens elicited Th1 dominant immunity in mice; PSMA, HPN, and AMACR. Each single antigen vaccine demonstrated significant anti-tumor activity inhibiting growth of implanted Myc-Cap cells after immunization as compared to control. Immunization with the combination of antigens, however, was superior to each alone in controlling tumor growth. When vaccination occurred simultaneously to tumor implant, multiantigen immunized mice had significantly smaller tumors than controls (p = 0.002) and a significantly improved overall survival (p = 0.0006). This multiantigen vaccine shows anti-tumor activity in a murine model of prostate cancer.

Calreticulin mutant myeloproliferative neoplasms induce MHC-I skewing, which can be overcome by an optimized peptide cancer vaccine.

The majority of JAK2V617F-negative myeloproliferative neoplasms (MPNs) have disease-initiating frameshift mutations in calreticulin (CALR), resulting in a common carboxyl-terminal mutant fragment (CALRMUT), representing an attractive source of neoantigens for cancer vaccines. However, studies have shown that CALRMUT-specific T cells are rare in patients with CALRMUT MPN for unknown reasons. We examined class I major histocompatibility complex (MHC-I) allele frequencies in patients with CALRMUT MPN from two independent cohorts. We observed that MHC-I alleles that present CALRMUT neoepitopes with high affinity are underrepresented in patients with CALRMUT MPN. We speculated that this was due to an increased chance of immune-mediated tumor rejection by individuals expressing one of these MHC-I alleles such that the disease never clinically manifested. As a consequence of this MHC-I allele restriction, we reasoned that patients with CALRMUT MPN would not efficiently respond to a CALRMUT fragment cancer vaccine but would when immunized with a modified CALRMUT heteroclitic peptide vaccine approach. We found that heteroclitic CALRMUT peptides specifically designed for the MHC-I alleles of patients with CALRMUT MPN efficiently elicited a CALRMUT cross-reactive CD8+ T cell response in human peripheral blood samples but not to the matched weakly immunogenic CALRMUT native peptides. We corroborated this effect in vivo in mice and observed that C57BL/6J mice can mount a CD8+ T cell response to the CALRMUT fragment upon immunization with a CALRMUT heteroclitic, but not native, peptide. Together, our data emphasize the therapeutic potential of heteroclitic peptide-based cancer vaccines in patients with CALRMUT MPN.

Cemeteries as sources of Aedes aegypti and other mosquito species in southeastern Puerto Rico.

OBJECTIVE: To investigate the presence and abundance of mosquito species in containers found in different types of cemeteries in Puerto Rico to assess their importance and make control recommendations. METHODS: We conducted surveys of containers with water in 16 cemeteries in southeastern Puerto Rico to detect the presence of larvae and pupae of Aedes aegypti and other mosquitoes; to identify the most common and productive containers and to study their variation in relation to the type of cemetery. RESULTS: The most common containers with water were flowerpots, followed in abundance by a variety of discarded containers and open tombs. We found a positive relationship between density of containers with water and rainfall. There was a rich community of mosquito species developing in containers of the inspected cemeteries: nine mosquito species belonging to four genera with Ae. aegypti and Ae. mediovittatus being the most frequent and abundant. We sampled 13 cement-type cemeteries, 2 mixed and only 1 lawn cemetery, consequently, we could not draw any conclusion regarding container productivity and cemetery type. CONCLUSIONS: Surveyed cemeteries were important sources of Ae. aegypti and other mosquitoes in flowerpots, discarded containers and open tombs. We recommend conducting further studies to establish how frequently inspections should occur; and mosquito control by emptying aquatic habitats and larviciding to reduce mosquito productivity and protect workers and visitors from mosquito bites and possible transmission of arboviruses.

Analysis of classical neutrophils and polymorphonuclear myeloid-derived suppressor cells in cancer patients and tumor-bearing mice.

In this study, using single-cell RNA-seq, cell mass spectrometry, flow cytometry, and functional analysis, we characterized the heterogeneity of polymorphonuclear neutrophils (PMNs) in cancer. We describe three populations of PMNs in tumor-bearing mice: classical PMNs, polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), and activated PMN-MDSCs with potent immune suppressive activity. In spleens of mice, PMN-MDSCs gradually replaced PMNs during tumor progression. Activated PMN-MDSCs were found only in tumors, where they were present at the very early stages of the disease. These populations of PMNs in mice could be separated based on the expression of CD14. In peripheral blood of cancer patients, we identified two distinct populations of PMNs with characteristics of classical PMNs and PMN-MDSCs. The gene signature of tumor PMN-MDSCs was similar to that in mouse activated PMN-MDSCs and was closely associated with negative clinical outcome in cancer patients. Thus, we provide evidence that PMN-MDSCs are a distinct population of PMNs with unique features and potential for selective targeting opportunities.

True umbilical cord knot, an emergency during labor.

An umbilical cord knot is an unexpected event that should not change obstetric approach for delivery.

Piomioma durante el puerperio: reporte de un caso / Pyomyoma in puerperium: a case report

Resumen ANTECEDENTES: La prevalencia de miomas durante el embarazo varía de 2-10%. Se calcula que 1 de cada 10 pacientes embarazadas tendrá complicaciones relacionadas con miomas (dolor, aumento del riesgo de aborto, parto pretérmino, posiciones fetales anómalas, entre otras). CASO CLÍNICO: Paciente primigesta de 30 años, con sepsis puerperal por piomioma, con febrícula como única manifestación clínica. El diagnóstico de piomioma se estableció después de descartar otras causas de fiebre en el puerperio y se confirmó con ecografía transvaginal y tomografía computada. El tratamiento consistió en histerectomía y antibióticos, con evolución favorable posterior. CONCLUSIÓN: El piomioma es una alteración excepcional durante el embrazo. La alta mortalidad refleja la necesidad del diagnóstico temprano, con la finalidad de iniciar el tratamiento con antibiótico y posterior intervención quirúrgica.

Abstract BACKGROUND: During pregnancy the incidence of myoma is from 2-10%. It is known that 1 of 10 cases will have problems related to the myoma (pain, pregnancy loss increased risk, preterm birth, anomal fetal position) CLINICAL CASE: 30-year-old primiparous patient, with puerperal sepsis due to piomyoma, with low-grade fever as the only clinical manifestation. The diagnosis of piomyoma was established after ruling out other causes of fever in the puerperium and was confirmed with transvaginal ultrasound and computed tomography. The treatment consisted of hysterectomy and antibiotic therapy, with favorable later evolution. CONCLUSIONS: The piomyoma is an exceptional alteration during the pregnancy. The high mortality reflects the need for early diagnosis, in order to initiate antibiotic therapy and subsequent surgical intervention.

Cancer-Associated Fibroblasts Neutralize the Anti-tumor Effect of CSF1 Receptor Blockade by Inducing PMN-MDSC Infiltration of Tumors.

Tumor-associated macrophages (TAM) contribute to all aspects of tumor progression. Use of CSF1R inhibitors to target TAM is therapeutically appealing, but has had very limited anti-tumor effects. Here, we have identified the mechanism that limited the effect of CSF1R targeted therapy. We demonstrated that carcinoma-associated fibroblasts (CAF) are major sources of chemokines that recruit granulocytes to tumors. CSF1 produced by tumor cells caused HDAC2-mediated downregulation of granulocyte-specific chemokine expression in CAF, which limited migration of these cells to tumors. Treatment with CSF1R inhibitors disrupted this crosstalk and triggered a profound increase in granulocyte recruitment to tumors. Combining CSF1R inhibitor with a CXCR2 antagonist blocked granulocyte infiltration of tumors and showed strong anti-tumor effects.

VISTA is an inhibitory immune checkpoint that is increased after ipilimumab therapy in patients with prostate cancer.

To date, anti-CTLA-4 (ipilimumab) or anti-PD-1 (nivolumab) monotherapy has not been demonstrated to be of substantial clinical benefit in patients with prostate cancer. To identify additional immune-inhibitory pathways in the prostate-tumor microenvironment, we evaluated untreated and ipilimumab-treated tumors from patients in a presurgical clinical trial. Levels of the PD-L1 and VISTA inhibitory molecules increased on independent subsets of macrophages in treated tumors. Our data suggest that VISTA represents another compensatory inhibitory pathway in prostate tumors after ipilimumab therapy.

The DNA methylation profile of activated human natural killer cells.

Natural killer (NK) cells are now recognized to exhibit characteristics akin to cells of the adaptive immune system. The generation of adaptive memory is linked to epigenetic reprogramming including alterations in DNA methylation. The study herein found reproducible genome wide DNA methylation changes associated with human NK cell activation. Activation led predominately to CpG hypomethylation (81% of significant loci). Bioinformatics analysis confirmed that non-coding and gene-associated differentially methylated sites (DMS) are enriched for immune related functions (i.e., immune cell activation). Known DNA methylation-regulated immune loci were also identified in activated NK cells (e.g., TNFA, LTA, IL13, CSF2). Twenty-one loci were designated high priority and further investigated as potential markers of NK activation. BHLHE40 was identified as a viable candidate for which a droplet digital PCR assay for demethylation was developed. The assay revealed high demethylation in activated NK cells and low demethylation in naïve NK, T- and B-cells. We conclude the NK cell methylome is plastic with potential for remodeling. The differentially methylated region signature of activated NKs revealed similarities with T cell activation, but also provided unique biomarker candidates of NK activation, which could be useful in epigenome-wide association studies to interrogate the role of NK subtypes in global methylation changes associated with exposures and/or disease states.

Lectin-type oxidized LDL receptor-1 distinguishes population of human polymorphonuclear myeloid-derived suppressor cells in cancer patients.

Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) are important regulators of immune responses in cancer and have been directly implicated in promotion of tumor progression. However, the heterogeneity of these cells and lack of distinct markers hampers the progress in understanding of the biology and clinical importance of these cells. Using partial enrichment of PMN-MDSC with gradient centrifugation we determined that low density PMN-MDSC and high density neutrophils from the same cancer patients had a distinct gene profile. Most prominent changes were observed in the expression of genes associated with endoplasmic reticulum (ER) stress. Surprisingly, low-density lipoprotein (LDL) was one of the most increased regulators and its receptor oxidized LDL receptor 1 OLR1 was one of the most overexpressed genes in PMN-MDSC. Lectin-type oxidized LDL receptor 1 (LOX-1) encoded by OLR1 was practically undetectable in neutrophils in peripheral blood of healthy donors, whereas 5-15% of total neutrophils in cancer patients and 15-50% of neutrophils in tumor tissues were LOX-1+. In contrast to their LOX-1- counterparts, LOX-1+ neutrophils had gene signature, potent immune suppressive activity, up-regulation of ER stress, and other biochemical characteristics of PMN-MDSC. Moreover, induction of ER stress in neutrophils from healthy donors up-regulated LOX-1 expression and converted these cells to suppressive PMN-MDSC. Thus, we identified a specific marker of human PMN-MDSC associated with ER stress and lipid metabolism, which provides new insight to the biology and potential therapeutic targeting of these cells.

High Resolution Viscosity Measurement by Thermal Noise Detection.

An interferometric method is implemented in order to accurately assess the thermal fluctuations of a micro-cantilever sensor in liquid environments. The power spectrum density (PSD) of thermal fluctuations together with Sader's model of the cantilever allow for the indirect measurement of the liquid viscosity with good accuracy. The good quality of the deflection signal and the characteristic low noise of the instrument allow for the detection and corrections of drawbacks due to both the cantilever shape irregularities and the uncertainties on the position of the laser spot at the fluctuating end of the cantilever. Variation of viscosity below 0:03mPa·s was detected with the alternative to achieve measurements with a volume as low as 50 µL.

Impact of Cell-surface Antigen Expression on Target Engagement and Function of an Epidermal Growth Factor Receptor × c-MET Bispecific Antibody.

The efficacy of engaging multiple drug targets using bispecific antibodies (BsAbs) is affected by the relative cell-surface protein levels of the respective targets. In this work, the receptor density values were correlated to the in vitro activity of a BsAb (JNJ-61186372) targeting epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (c-MET). Simultaneous binding of the BsAb to both receptors was confirmed in vitro. By using controlled Fab-arm exchange, a set of BsAbs targeting EGFR and c-MET was generated to establish an accurate receptor quantitation of a panel of lung and gastric cancer cell lines expressing heterogeneous levels of EGFR and c-MET. EGFR and c-MET receptor density levels were correlated to the respective gene expression levels as well as to the respective receptor phosphorylation inhibition values. We observed a bias in BsAb binding toward the more highly expressed of the two receptors, EGFR or c-MET, which resulted in the enhanced in vitro potency of JNJ-61186372 against the less highly expressed target. On the basis of these observations, we propose an avidity model of how JNJ-61186372 engages EGFR and c-MET with potentially broad implications for bispecific drug efficacy and design.

Engagement of the ICOS pathway markedly enhances efficacy of CTLA-4 blockade in cancer immunotherapy.

Cytotoxic T lymphocyte antigen-4 (CTLA-4) blockade with a monoclonal antibody yields durable responses in a subset of cancer patients and has been approved by the FDA as a standard therapy for late-stage melanoma. We recently identified inducible co-stimulator (ICOS) as a crucial player in the antitumor effects of CTLA-4 blockade. We now show that concomitant CTLA-4 blockade and ICOS engagement by tumor cell vaccines engineered to express ICOS ligand enhanced antitumor immune responses in both quantity and quality and significantly improved rejection of established melanoma and prostate cancer in mice. This study provides strong support for the development of combinatorial therapies incorporating anti-CTLA-4 and ICOS engagement.

Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma.

Treatment with monoclonal antibody specific for cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), an inhibitory receptor expressed by T lymphocytes, has emerged as an effective therapy for the treatment of metastatic melanoma. Although subject to debate, current models favor a mechanism of activity involving blockade of the inhibitory activity of CTLA-4 on both effector (T eff) and regulatory (T reg) T cells, resulting in enhanced antitumor effector T cell activity capable of inducing tumor regression. We demonstrate, however, that the activity of anti-CTLA-4 antibody on the T reg cell compartment is mediated via selective depletion of T reg cells within tumor lesions. Importantly, T reg cell depletion is dependent on the presence of Fcγ receptor-expressing macrophages within the tumor microenvironment, indicating that T reg cells are depleted in trans in a context-dependent manner. Our results reveal further mechanistic insight into the activity of anti-CTLA-4-based cancer immunotherapy, and illustrate the importance of specific features of the local tumor environment on the final outcome of antibody-based immunomodulatory therapies.

Cutting edge: chronic inflammatory liver disease in mice expressing a CD28-specific ligand.

Inflammation of the normally tolerant liver microenvironment precedes the development of chronic liver disease. Study of the pathogenesis of autoimmune liver diseases, such as autoimmune hepatitis (AIH), has been hampered by a lack of autochthonous chronic animal models. Through our studies of T cell costimulation, we generated transgenic mice expressing a ligand specific for the CD28 receptor, which normally shares ligands with the related inhibitory receptor CTLA-4. The mice spontaneously develop chronic inflammatory liver disease with several pathologies found in AIH, including elevated serum aminotransferases in the context of normal alkaline phosphatase and bilirubin levels, lymphocytic inflammation, focal necrosis, oval cell hyperplasia, and fibrosis. The prevalence of IFN-γ-producing CD8(+) T cells in the livers of transgenic mice suggests a role for autoimmune cytotoxicity in the chronic disease state. The CD28 ligand-specific transgenic mice will facilitate evaluation of CD8(+) T cell function in liver disease pathologies found in AIH.

Factores asociados a consumo de tabaco durante el último año en estudiantes de educación superior / Smoking associated factors in students during their last year of higher education / Fatores associados a consumo do cigarro durante o último ano em estudantes de educação superior

Objetivo. Determinar los factores de riesgo asociados al consumo de tabaco en el último año en una población universitaria chilena. Metodología. Estudio observacional analítico, de corte transversal. Se realizó muestreo aleatorio estratificado según unidad académica y año de carrera, siendo el tamaño muestral de 2045 estudiantes de cuatro Campus de la Universidad Católica de Chile. La toma de la información se hizo con ayuda de un cuestionario autoaplicado. Se realizaron análisis descriptivo, bivariado y de regresión logística. Resultados. El promedio de edad fue 20.4±1,6 años, el 46.6% eran de sexo masculino y el70.3% tenían nivel socioeconómico alto. Un 91.0% había iniciado el consumo después de los 18 años. La prevalencia mensual de consumo de tabaco fue de 40.5%, un 20.0% de los encuestados estaría a riesgo de tener diagnóstico de tabaquismo. Se encontró asociación positiva entre consumo de tabaco durante último año y: la edad (p<0.05), el sexo femenino (p<0.001), el nivel socioeconómico (p<0.05) y la percepción de consumo de droga por parte de padres (p<0.001). Se encontró asociación negativa entre consumo de tabaco y la religiosidad (p<0.001).Conclusión. En el grupo universitario en el que se realizó el estudio se encontró que el problema de tabaquismo merece especial atención por su magnitud, estando asociado a factores de riesgo como la edad, el sexo femenino, el estrato socioeconómico y una baja religiosidad.

Objective. To determine the risk factors associated to smokingin the last year of school in a Chilean university population. Methodology. Analytical, observational, transversal cut study. Stratified randomized sampling was conducted according to academic unit and year of career; the size of the sample was of 2045 students from four campuses of the catholic university of Chile. The information was collected with a self-applied questionnaire. A descriptive, bivariate and logistic regression analysis was made. Results. The average age was 20.4±1.6,46% were males and 70.3% had a high socioeconomic status. 91% had started cigarette consumption before the age of 18. The smoking monthly prevalence was of 40.5%, 20% of the surveyed would be atrisk of having diagnosis of smoking. A positive association was found between smoking during the last year of university and: age (p<0.05), female sex (p<0.001), socioeconomic status (p<0.05) and parents drugconsumption perception (p<0.001). A negative association was found between smoking and religiosity(p<0.001). Conclusion. In the studied university group it was found that the smoking problem requires special attention due to its magnitude and association to risk factors as age, female gender, socioeconomic status and a low religiosity.

Objetivo. Determinar os fatores de risco associados ao consumo do cigarro no último ano numa população universitária chilena. Metodologia. Estudo observacional analítico, de corte transversal. Realizou-se amostra gemaleatória estratificado segundo unidade acadêmica e ano de carreira, sendo o tamanho da mostra de2 045 estudantes de quatro Campus da Universidade Católica de Chile. A tomada da informação se fez comajuda de um questionário auto-aplicado. Realizaram-se análise descritiva, bivariado e de regressão logística. Resultados. A média de idade foi 20.4±1,6 anos, o 46.6% eram de sexo masculino e o 70.3% tinham nível socioeconômico alto. Um 91% tinha iniciado o consumo depois dos 18 anos. A prevalência mensal de consumo do cigarro foi de 40.5%, um 20% dos interrogados estaria a risco de ter diagnóstico de tabagismo. Encontrou-se associação positiva entre consumo de fumo durante último ano e: a idade (p<0.05), o sexo feminino (p<0.001), o nível socioeconômico (p<0.05) e a percepção de consumo de droga por parte de pais (p<0.001). Encontrou-se associação negativa entre consumo de fumo e a religiosidade (p<0.001). Conclusão. No grupo universitário no que se realizou o estudo se encontrou que o problema de tabagismo merece especial atendimento por sua magnitude, estando sócio a fatores de risco como a idade, o sexo feminino,o estrato socioeconômico e uma baixa religiosidade.

Propensity score: una aplicación en medicina / Propensity score: a medical aplication

En cualquier investigación en que queremos comparar el efecto de dos o más “tratamientos” sobre una “respuesta” de interés nos resultaría ideal asignar aleatoriamente los “sujetos” a los tratamientos, con la esperanza de que procediendo así controlaríamos la presencia (y distribución) de factores que pueden ser influyentes en el nivel de la respuesta, distorsionando, en una forma desconocida, la influencia real de los tratamientos. Muchas veces tal aleatorización simplemente es impensable por razones éticas o prácticas. Una solución adecuada en muchas investigaciones médicas, sociológicas, psicológicas o económicas ha sido el uso del Propensity Score, que se presenta en este artículo ilustrándolo con una aplicación médica.

In any research in which we want to compare the effect of two or more"treatments" on a "response" of interest, it would be ideal to randomlyassign "subjects" to treatment, hoping that by doing so we will control the presence (and distribution ) of factors that may be influential on the response leve distorting, in an unknown way, the real influence of the treatments. Often, such randomization, simplermente is unthinkable for ethical or operational reasons. A good solution in many medical, sociological, psychological or economic research has been the use of the Propensity Score presented in this article along with medical application.

Changes in replication, nuclear location, and expression of the Igh locus after fusion of a pre-B cell line with a T cell line.

We have previously observed that replication and nuclear location of the murine Igh locus are developmentally regulated during B cell differentiation. In non-B, B, and plasma cells, sequences near the 3' end of the Igh locus replicate early in S while upstream Vh sequences replicate late in S, and the Igh locus is located near the nuclear periphery. In fact, in MEL non-B cells, replication of a 500-kb segment containing Igh-C and flanking sequences occurs progressively later throughout S by 3' to 5' unidirectional fork movement. In contrast, in pro- and pre-B cells, the entire 3-Mb Igh locus is located away from the nuclear periphery and replicates early in S by forks progressing in both directions. In this study, using an 18-81 (pre-B) x BW5147 (T) cell fusion system in which Igh expression is extinguished, we found that in all Igh alleles, Vh sequences replicated later in S than 3' Igh sequences (similar to that detected in BW5147), but the Igh locus was situated away from the nuclear periphery (similar to that observed in 18-81). Thus, pre-B cell-derived Igh genes had changes in replication timing, but not in nuclear location, whereas T cell-derived Igh genes changed their nuclear location but not their replication timing. These data are consistent with the silencing of a pre-B cell-specific replication program in the fusion hybrid cells and independent regulation of the nuclear location of Igh loci.

Chromatin architecture near a potential 3' end of the igh locus involves modular regulation of histone modifications during B-Cell development and in vivo occupancy at CTCF sites.

The murine Igh locus has a 3' regulatory region (3' RR) containing four enhancers (hs3A, hs1,2, hs3B, and hs4) at DNase I-hypersensitive sites. The 3' RR exerts long-range effects on class switch recombination (CSR) to several isotypes through its control of germ line transcription. By measuring levels of acetylated histones H3 and H4 and of dimethylated H3 (K4) with chromatin immunoprecipitation assays, we found that early in B-cell development, chromatin encompassing the enhancers of the 3' RR began to attain stepwise modifications typical of an open conformation. The hs4 enhancer was associated with active chromatin initially in pro- and pre-B cells and then together with hs3A, hs1,2, and hs3B in B and plasma cells. Histone modifications were similar in resting splenic B cells and in splenic B cells induced by lipopolysaccharide to undergo CSR. From the pro-B-cell stage onward, the approximately 11-kb region immediately downstream of hs4 displayed H3 and H4 modifications indicative of open chromatin. This region contained newly identified DNase I-hypersensitive sites and several CTCF target sites, some of which were occupied in vivo in a developmentally regulated manner. The open chromatin environment of the extended 3' RR in mature B cells was flanked by regions associated with dimethylated K9 of histone H3. Together, these data suggest that 3' RR elements are located within a specific chromatin subdomain that contains CTCF binding sites and developmentally regulated modules.