Interferon-gamma is quintessential for NOS2 and COX2 expression in ER- breast tumors that lead to poor outcome.

A strong correlation between NOS2 and COX2 tumor expression and poor clinical outcomes in ER breast cancer has been established. However, the mechanisms of tumor induction of these enzymes are unclear. Analysis of The Cancer Genome Atlas (TCGA) revealed correlations between NOS2 and COX2 expression and Th1 cytokines. Herein, single-cell RNAseq analysis of TNBC cells shows potent NOS2 and COX2 induction by IFNγ combined with IL1ß or TNFα. Given that IFNγ is secreted by cytolytic lymphocytes, which improve clinical outcomes, this role of IFNγ presents a dichotomy. To explore this conundrum, tumor NOS2, COX2, and CD8+ T cells were spatially analyzed in aggressive ER-, TNBC, and HER2 + breast tumors. High expression and clustering of NOS2-expressing tumor cells occurred at the tumor/stroma interface in the presence of stroma-restricted CD8+ T cells. High expression and clustering of COX2-expressing tumor cells extended into immune desert regions in the tumor core where CD8+ T cell penetration was limited or absent. Moreover, high NOS2-expressing tumor cells were proximal to areas with increased satellitosis, suggestive of cell clusters with a higher metastatic potential. Further in vitro experiments revealed that IFNγ + IL1ß/TNFα increased the elongation and migration of treated tumor cells. This spatial analysis of the tumor microenvironment provides important insight into distinct neighborhoods where stroma-restricted CD8+ T cells exist proximal to NOS2-expressing tumor niches that could have increased metastatic potential.

Interferon-gamma is Quintessential for NOS2 and COX2 Expression in ER - Breast Tumors that Lead to Poor Outcome.

A strong correlation between NOS2 and COX2 tumor expression and poor clinical outcomes in ER-breast cancer has been established. However, mechanisms of tumor induction of these enzymes are unclear. Analysis of The Cancer Genome Atlas (TCGA) revealed correlations between NOS2 and COX2 expression and Th1 cytokines. Herein, single cell RNAseq analysis of TNBC cells shows potent NOS2 and COX2 induction by IFNγ combined with IL1ß or TNFα. Given that IFNγ is secreted by cytolytic lymphocytes, which improve clinical outcomes, this role of IFNγpresents a dichotomy. To explore this conundrum, tumor NOS2, COX2, and CD8 + T cells were spatially analyzed in aggressive ER-, TNBC, and HER2+ breast tumors. High expression and clustering of NOS2-expressing tumor cells occurred at the tumor/stroma interface in the presence of stroma-restricted CD8 + T cells. High expression and clustering of COX2-expressing tumor cells extended into immune desert regions in the tumor core where CD8 + T cell penetration was limited or absent. Moreover, high NOS2-expressing tumor cells were proximal to areas with increased satellitosis suggestive of cell clusters with a higher metastatic potential. Further in vitro experiments revealed that IFNγ+IL1ß/TNFα increased elongation and migration of treated tumor cells. This spatial analysis of the tumor microenvironment provides important insight of distinct neighborhoods where stroma-restricted CD8 + T cells exist proximal to NOS2-expressing tumor niches that could have increased metastatic potential.

NOS2 and COX2 Provide Key Spatial Targets that Determine Outcome in ER- Breast Cancer.

Estrogen receptor-negative (ER-) breast cancer is an aggressive breast cancer subtype with limited therapeutic options. Upregulated expression of both inducible nitric oxide synthase (NOS2) and cyclo-oxygenase (COX2) in breast tumors predicts poor clinical outcomes. Signaling molecules released by these enzymes activate oncogenic pathways, driving cancer stemness, metastasis, and immune suppression. The influence of tumor NOS2/COX2 expression on the landscape of immune markers using multiplex fluorescence imaging of 21 ER- breast tumors were stratified for survival. A powerful relationship between tumor NOS2/COX2 expression and distinct CD8+ T cell phenotypes was observed at 5 years post-diagnosis. These results were confirmed in a validation cohort using gene expression data showing that ratios of NOS2 to CD8 and COX2 to CD8 are strongly associated with poor outcomes in high NOS2/COX2-expressing tumors. Importantly, multiplex imaging identified distinct CD8+ T cell phenotypes relative to tumor NOS2/COX2 expression in Deceased vs Alive patient tumors at 5-year survival. CD8+NOS2-COX2- phenotypes defined fully inflamed tumors with significantly elevated CD8+ T cell infiltration in Alive tumors expressing low NOS2/COX2. In contrast, two distinct phenotypes including inflamed CD8+NOS2+COX2+ regions with stroma-restricted CD8+ T cells and CD8-NOS2-COX2+ immune desert regions with abated CD8+ T cell penetration, were significantly elevated in Deceased tumors with high NOS2/COX2 expression. These results were supported by applying an unsupervised nonlinear dimensionality-reduction technique, UMAP, correlating specific spatial CD8/NOS2/COX2 expression patterns with patient survival. Moreover, spatial analysis of the CD44v6 and EpCAM cancer stem cell (CSC) markers within the CD8/NOS2/COX2 expression landscape revealed positive correlations between EpCAM and inflamed stroma-restricted CD8+NOS2+COX2+ phenotypes at the tumor/stroma interface in deceased patients. Also, positive correlations between CD44v6 and COX2 were identified in immune desert regions in deceased patients. Furthermore, migrating tumor cells were shown to occur only in the CD8-NOS2+COX2+ regions, identifying a metastatic hot spot. Taken together, this study shows the strength of spatial localization analyses of the CD8/NOS2/COX2 landscape, how it shapes the tumor immune microenvironment and the selection of aggressive tumor phenotypes in distinct regions that lead to poor clinical outcomes. This technique could be beneficial for describing tumor niches with increased aggressiveness that may respond to clinically available NOS2/COX2 inhibitors or immune-modulatory agents.

Spatial analysis of NOS2 and COX2 interaction with T-effector cells reveals immunosuppressive landscapes associated with poor outcome in ER- breast cancer patients.

Multiple immunosuppressive mechanisms exist in the tumor microenvironment that drive poor outcomes and decrease treatment efficacy. The co-expression of NOS2 and COX2 is a strong predictor of poor prognosis in ER- breast cancer and other malignancies. Together, they generate pro-oncogenic signals that drive metastasis, drug resistance, cancer stemness, and immune suppression. Using an ER- breast cancer patient cohort, we found that the spatial expression patterns of NOS2 and COX2 with CD3+CD8+PD1- T effector (Teff) cells formed a tumor immune landscape that correlated with poor outcome. NOS2 was primarily associated with the tumor-immune interface, whereas COX2 was associated with immune desert regions of the tumor lacking Teff cells. A higher ratio of NOS2 or COX2 to Teff was highly correlated with poor outcomes. Spatial analysis revealed that regional clustering of NOS2 and COX2 was associated with stromal-restricted Teff, while only COX2 was predominant in immune deserts. Examination of other immunosuppressive elements, such as PDL1/PD1, Treg, B7H4, and IDO1, revealed that PDL1/PD1, Treg, and IDO1 were primarily associated with restricted Teff, whereas B7H4 and COX2 were found in tumor immune deserts. Regardless of the survival outcome, other leukocytes, such as CD4 T cells and macrophages, were primarily in stromal lymphoid aggregates. Finally, in a 4T1 model, COX2 inhibition led to a massive cell infiltration, thus validating the hypothesis that COX2 is an essential component of the Teff exclusion process and, thus, tumor evasion. Our study indicates that NOS2/COX2 expression plays a central role in tumor immunosuppression. Our findings indicate that new strategies combining clinically available NOS2/COX2 inhibitors with various forms of immune therapy may open a new avenue for the treatment of aggressive ER-breast cancers.

Systemic Nos2 Depletion and Cox inhibition limits TNBC disease progression and alters lymphoid cell spatial orientation and density.

Antitumor immune polarization is a key predictor of clinical outcomes to cancer therapy. An emerging concept influencing clinical outcome involves the spatial location of CD8+ T cells, within the tumor. Our earlier work demonstrated immunosuppressive effects of NOS2 and COX2 tumor expression. Here, we show that NOS2/COX2 levels influence both the polarization and spatial location of lymphoid cells including CD8+ T cells. Importantly, elevated tumor NOS2/COX2 correlated with exclusion of CD8+ T cells from the tumor epithelium. In contrast, tumors expressing low NOS2/COX2 had increased CD8+ T cell penetration into the tumor epithelium. Consistent with a causative relationship between these observations, pharmacological inhibition of COX2 with indomethacin dramatically reduced tumor growth of the 4T1 model of TNBC in both WT and Nos2- mice. This regimen led to complete tumor regression in ∼20-25% of tumor-bearing Nos2- mice, and these animals were resistant to tumor rechallenge. Th1 cytokines were elevated in the blood of treated mice and intratumoral CD4+ and CD8+ T cells were higher in mice that received indomethacin when compared to control untreated mice. Multiplex immunofluorescence imaging confirmed our phenotyping results and demonstrated that targeted Nos2/Cox2 blockade improved CD8+ T cell penetration into the 4T1 tumor core. These findings are consistent with our observations in low NOS2/COX2 expressing breast tumors proving that COX2 activity is responsible for limiting the spatial distribution of effector T cells in TNBC. Together these results suggest that clinically available NSAID's may provide a cost-effective, novel immunotherapeutic approach for treatment of aggressive tumors including triple negative breast cancer.

Child Protective Services Reports in the Context of Intimate Partner Violence: A Delphi Process Examining Best Practices.

OBJECTIVES: Child protective services (CPS) reporting for families experiencing intimate partner violence (IPV) is complex. The goal of this study was to develop expert-driven best practices for pediatric providers filing CPS reports in the context of IPV. METHODS: We conducted a Delphi study with experts in IPV and child abuse and neglect (CAN) through 3 rounds of surveys. In Round 1, participants selected clinical scenarios for which they would file, as well as best practices when CPS reporting is indicating. In Round 2, participants described how strongly they agreed that a provider should file for each clinical scenario and how important each best practice was on a 5-point Likert scale. Finally, in Round 3 participants reviewed Round 1 and 2 results, then reported their final determination by selecting yes or no for each option. Consensus was achieved in Round 3 if >80% of participants agreed. In each round, participants could provide further detail via free-text answers. RESULTS: Twenty-three (40%) of the invited experts participated. Consensus was not achieved for children directly witnessing IPV or experiencing health symptoms due to IPV exposure. Participants were in consensus regarding need for CPS reporting when CAN was present and that reporting should not occur for exposure to IPV only. Best practices included supporting IPV survivors, developing healthcare-based IPV advocacy programs, and optimizing the child welfare system. CONCLUSION: This study provides expert-driven recommendations for filing CPS reports in the context of IPV and highlights the inherent complexity of filing and the need for further guidelines.

Injuries Suggestive of Physical Abuse in Young Children With Subconjunctival Hemorrhages.

OBJECTIVE: The aim of this study was to compare the demographic characteristics, clinical presentations, medical evaluation, and injuries identified in a cohort of children with and without subconjunctival hemorrhage who were evaluated by a child abuse specialist. METHODS: This was a case-control study that used data from the ExSTRA (Examining Siblings to Recognize Abuse) research network. Subjects with a subconjunctival hemorrhage(s) were designated as cases. Four controls matched for age and participating center were included for each case. Descriptive statistics were used to compare cases and controls. RESULTS: Fifty of the 2890 subjects in the parent study had a subconjunctival hemorrhage(s) and were designated as cases. The cases had a median (interquartile range) age of 5.0 months (2.0-23.6 months). Two hundred controls were matched to the cases. There was no difference in the demographics, clinical characteristics, medical evaluation, or rate of occult injuries identified in cases and controls. Almost one-quarter of children with subconjunctival hemorrhages had no other external sign of trauma but had the same rate of occult injuries as children with bruises. CONCLUSIONS: These data suggest that subconjunctival hemorrhages are relatively rare among children undergoing evaluation by a child abuse specialist, but that they are often an indicator of occult injury. Even in the absence of other external signs of trauma, the presence of subconjunctival hemorrhages should prompt an age-appropriate evaluation for physical abuse.

Determinants of COVID-19 vaccine hesitancy in French hospitals.

OBJECTIVES: COVID-19 vaccines have become the new hope for stemming the pandemic. We aimed to assess pre-launch vaccine acceptance among hospital workers in the Auvergne-Rhône-Alpes Administrative Region of France. METHODS: We performed a cross sectional study involving all hospital workers in 11 Auvergne-Rhône-Alpes hospitals in December 2020. Univariate and multivariate analyses were performed to identify factors associated with vaccine hesitancy. RESULTS: We analyzed completed questionnaires from 1,964 respondents (78% women, mean age 42 years, 21.5% physicians, 41% private care centers). A total of 1,048 (53%) hospital workers were in favor of COVID-19 vaccination. Vaccine hesitancy was associated with: female gender; young age; paramedical, technical, and administrative professions (i.e., all non-medical professions); no prior flu vaccination; and employment in the private medical care sector (p<0.05). Distrust of health authorities and pharmaceutical lobbying were the main obstacles to vaccination. Inversely, creating herd immunity and protecting patients and household members were the most frequently cited reasons in favor of vaccination. More than two-thirds of participants feared that the clinical and biological research was too rapid and worried about serious adverse effects. Most participants were interested in written information on the available vaccines, but the most vaccine-hesitant categories preferred oral information. Only 35% supported mandatory vaccination. CONCLUSIONS: Targeted written and oral information campaigns will be necessary to improve vaccination coverage among hospital workers who show a surprisingly high hesitancy rate. Imposing mandatory vaccination could be counterproductive.

Posaconazole-induced hypertension in children with cystic fibrosis.

Posaconazole is a triazole antifungal with a broad spectrum of activity against moulds including Aspergillus spp. Emerging data suggest posaconazole may be effective in the treatment of allergic bronchopulmonary aspergillosis (ABPA) complicating cystic fibrosis (CF). Rarely, posaconazole can cause pseudohyperaldosteronism, manifesting as hypertension and electrolyte abnormalities, with a number of cases recently reported in individuals without CF. We describe two cases of children with CF who developed hypertension, likely due to pseudohyperaldosteronism, following the initiation of posaconazole for the treatment of ABPA.

"It Can Be Hard But It's Not Bad": Three Questions to Solicit Caregiver Perceptions of Benefits and Burdens to Participating in Pediatric Palliative Care Research.

Background: To allay uneasiness among clinicians and institutional review board members about pediatric palliative care research and to yield new knowledge relevant to study methods, documenting burdens and benefits of this research on children and their families is essential. Design: In a grounded theory study with three data points (T1, T2, and T3), we evaluated benefits and burdens of family caregiver participation at T3. English-speaking caregivers participating in palliative or end-of-life decisions for their child with incurable cancer or their seriously ill child in the intensive care unit participated. Thirty-seven caregivers (n = 22 from oncology; n = 15 from intensive care) of 33 children completed T3 interviews; most were mothers (n = 25, 67.6%), African American (n = 18, 48.6%), and married (n = 28, 75.7%). Measurement: Benefits and burdens were assessed by three open-ended questions asked by an interviewer during a scheduled telephone contact. Responses were analyzed using descriptive semantic content analysis techniques and themes were extracted. Results: All 37 T3 participants completed the 3 questions, resulting in no missing data. The most frequently reported themes were of positive personal impact: "Hoping to help others," "Speaking about what is hard is important," and "Being in the study was sometimes hard but not bad." Conclusions: No caregiver described the study as burdensome. Some acknowledged that answering the questions could evoke sad memories, but highlighted benefits for self and others. Attrition somewhat tempers the emphasis on benefits. Documenting perceived benefits and burdens in a standardized manner may accurately convey impact of study participation and yield new knowledge.

The 4D Space-Time Dimensions of Facial Perception.

Facial information is a powerful channel for human-to-human communication. Characteristically, faces can be defined as biological objects that are four-dimensional (4D) patterns, whereby they have concurrently a spatial structure and surface as well as temporal dynamics. The spatial characteristics of facial objects contain a volume and surface in three dimensions (3D), namely breadth, height and importantly, depth. The temporal properties of facial objects are defined by how a 3D facial structure and surface evolves dynamically over time; where time is referred to as the fourth dimension (4D). Our entire perception of another's face, whether it be social, affective or cognitive perceptions, is therefore built on a combination of 3D and 4D visual cues. Counterintuitively, over the past few decades of experimental research in psychology, facial stimuli have largely been captured, reproduced and presented to participants with two dimensions (2D), while remaining largely static. The following review aims to advance and update facial researchers, on the recent revolution in computer-generated, realistic 4D facial models produced from real-life human subjects. We delve in-depth to summarize recent studies which have utilized facial stimuli that possess 3D structural and surface cues (geometry, surface and depth) and 4D temporal cues (3D structure + dynamic viewpoint and movement). In sum, we have found that higher-order perceptions such as identity, gender, ethnicity, emotion and personality, are critically influenced by 4D characteristics. In future, it is recommended that facial stimuli incorporate the 4D space-time perspective with the proposed time-resolved methods.

Outpatient parenteral antibiotic therapy: Evaluation of practices and limits of use in rural areas in France.

OBJECTIVES: To evaluate outpatient parenteral antibiotic therapy (OPAT) practices in a French rural area. MATERIAL AND METHODS: Descriptive study assessing knowledge, practices, and limitations of OPAT use among hospital practitioners (HP), family physicians (FP), and private nurses (PN). RESULTS: OPAT (mainly ceftriaxone and penicillins) was used by 69.6%, 73.3%, and 97.7% of the 23 HPs, 45 FPs, and 46 PNs mostly for respiratory or urinary tract infections, bacteremia, and/or multidrug-resistant bacterial infections. Overall, 65.2% of HPs and 37.8% of FPs were in contact with an infectious disease specialist. Knowledge of OPAT benefits and risks was lower for FPs than HPs. The main obstacles were the patient's geographic isolation (HPs), the availability of a venous catheter, the lack of training (FPs), and the expected OPAT-associated overwork (PNs). CONCLUSION: OPAT practice is weak in rural areas. Declared obstacles constitute fields of improvement for its essential expansion.

Validation of the Functional Assessment of Chronic Illness Therapy-Spiritual Well-Being-Expanded (FACIT-Sp-Ex) Across English and Spanish-Speaking Hispanics/Latinos: Results From the Hispanic Community Health Study/Study of Latinos Sociocultural Ancillary Study.

The validity of the Functional Assessment of Chronic Illness Therapy-Spiritual Well-Being (FACIT-Sp) has been examined in primarily non-Hispanics/Latinos with chronic illness. This study assessed the psychometric properties of the non-illness, expanded FACIT-Sp (FACIT-Sp-Ex) in 5,163 U.S. Hispanic/Latino adults. Measures were interviewer-administered in English or Spanish. Confirmatory factor analyses indicated four factors: Meaning, Peace, Faith, and Relational. The scale demonstrated measurement invariance across English and Spanish. Subscales displayed adequate internal and test-retest reliability. Scores were positively associated with Duke Religion Index (DUREL) subscales. When all subscales were entered in a single model, Meaning and Peace were inversely associated with depressive symptoms and positively associated with HRQOL. Faith was positively associated with depressive symptoms and inversely associated with HRQOL. Relational was not associated with any outcome. FACIT-Sp-Ex subscales were generally more strongly associated than DUREL subscales with well-being. The FACIT-Sp-Ex appears to be a valid measure of spiritual well-being in U.S. Hispanics/Latinos.