Methods for Imaging Inflammation and Transendothelial Migration in Vivo and ex Vivo.

Inflammation is the body's response to injury and harmful stimuli and contributes to a range of infectious and noninfectious diseases. Inflammation occurs through a series of well-defined leukocyte-endothelial cell interactions, including rolling, activation, adhesion, transmigration, and subsequent migration through the extracellular matrix. Being able to visualize the stages of inflammation is important for a better understanding of its role in diseases processes. Detailed in this article are protocols for imaging immune cell infiltration and transendothelial migration in vascular tissue beds, including those in the mouse ear, cremaster muscle, brain, lung, and retina. Also described are protocols for inducing inflammation and quantifying leukocytes with FIJI imaging software. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Induction of croton oil dermatitis Alternate Protocol 1: Induction of croton oil dermatitis using genetically fluorescent mice Basic Protocol 2: Intravital microscopy of the mouse cremaster muscle Support Protocol: Making a silicone stage Basic Protocol 3: Wide-field microscopy of the mouse brain Basic Protocol 4: Imaging the lungs (ex vivo) Alternate Protocol 2: Inflating the lungs without tracheostomy Basic Protocol 5: Inducing, imaging, and quantifying infiltration of leukocytes in mouse retina.

Platelet Endothelial Cell Adhesion Molecule (PECAM/CD31) Blockade Modulates Neutrophil Recruitment Patterns and Reduces Infarct Size in Experimental Ischemic Stroke.

The infiltration of polymorphonuclear leukocytes (PMNs) in ischemia-reperfusion injury (I/RI) has been implicated as a critical component of inflammatory damage following ischemic stroke. However, successful blockade of PMN transendothelial migration (TEM) in preclinical studies has not translated to meaningful clinical outcomes. To investigate this further, leukocyte infiltration patterns were quantified, and these patterns were modulated by blocking platelet endothelial cell adhesion molecule-1 (PECAM), a key regulator of TEM. LysM-eGFP mice and microscopy were used to visualize all myeloid leukocyte recruitment following ischemia/reperfusion. Visual examination showed heterogeneous leukocyte distribution across the infarct at both 24 and 72 hours after I/RI. A semiautomated process was designed to precisely map PMN position across brain sections. Treatment with PECAM function-blocking antibodies did not significantly affect total leukocyte recruitment but did alter their distribution, with more observed at the cortex at both early and later time points (24 hours: 89% PECAM blocked vs. 72% control; 72 hours: 69% PECAM blocked vs. 51% control). This correlated with a decrease in infarct volume. These findings suggest that TEM, in the setting of I/RI in the cerebrovasculature, occurs primarily at the cortical surface. The reduction of stroke size with PECAM blockade suggests that infiltrating PMNs may exacerbate I/RI and indicate the potential therapeutic benefit of regulating the timing and pattern of leukocyte infiltration after stroke.

Inhibition of PECAM-1 Significantly Delays Leukocyte Extravasation into the Subcortex Post-Stroke.

BACKGROUND AND PURPOSE: Current therapies for ischemic stroke focus on reperfusion but do not address the acute inflammatory response that results in significant reperfusion injury. To advance future therapies, a thorough understanding of the precise spatiotemporal underpinnings of leukocyte extravasation and infiltration is necessary. We describe the evolution of the inflammatory response in a mouse transient middle cerebral artery occlusion (tMCAO) stroke model at several time points after reperfusion and the modulation of this response with PECAM blockade. METHODS: The transient Middle Cerebral Artery Occlusion model (90 minutes of ischemia followed by reperfusion) was used to simulate large vessel occlusion stroke and recanalization. We used wide field and confocal immunofluorescence microscopy to examine the exact distribution of neutrophils with close examination of the leukocyte position with regard to the brain vasculature and the perivascular space. Flow cytometry of single cell suspensions was used to confirm cell identity at different time points post-stroke. RESULTS: Large ischemic strokes involving both the subcortex and cortex (over 20% of the ischemic hemisphere) were induced in mice. At 12 and 24 hours, leukocyte recruitment and extravasation was primarily localized to the cortical surface. This contrasts with other organs where there is considerable migration of neutrophils deep into the inflamed tissue by 24 hours. Flow cytometry showed at 24 hours a majority of leukocytes were neutrophils. Over 48 to 72 hours, leukocytes were increasingly found deeper into the subcortex. Throughout the infarct (determined with triphenyl tetrazolium chloride staining), leukocyte recruitment was not uniform but rather organized in clusters. Disrupting leukocyte diapedesis with PECAM function-blocking monoclonal antibody restricted leukocytes to within 500 microns of the surface when compared to control; and this was still evident at 72 hours (n=3 mice per group, p<0.01, Control 46% ± 4.0 %; PECAM-1 Ab 62% ± 5.0%). High-resolution wide-field microscopy confirmed inhibition of TEM by PECAM-1 blockade at 24 hours. Flow cytometry showed approximately equal numbers of monocytes and neutrophils at 72 hours. CONCLUSIONS: Our findings demonstrate that leukocyte infiltration into a stroke evolves over several days following reperfusion. The use of PECAM blockade modulates the natural progression of leukocytes into the infarcted stroke bed. A better understanding of leukocyte spatiotemporal infiltration and its regulators could help inform the next generation of therapeutic interventions.

Author Correction: High-fat diet enhances stemness and tumorigenicity of intestinal progenitors.

In Fig. 4e of this Article, the labels for 'Control' and 'HFD' were reversed ('Control' should have been labelled blue rather than purple, and 'HFD' should have been labelled purple rather than blue). Similarly, in Fig. 4f of this Article, the labels for 'V' and 'GW' were reversed ('V' should have been labelled blue rather than purple, and 'GW' should have been labelled purple instead of blue). The original figure has been corrected online.

Land Reuse Site Screening Tool Cohorts: Creating Land Reuse Site Inventories.

As part of our continued effort to highlight innovative approaches to improve the health and environment of communities, the Journal is pleased to publish a bimonthly column from the Agency for Toxic Substances and Disease Registry (ATSDR) at the Centers for Disease Control and Prevention (CDC). ATSDR serves the public by using the best science, taking responsive public health actions, and providing trusted health information to prevent harmful exposures and diseases related to toxic substances. The purpose of this column is to inform readers of ATSDR's activities and initiatives to better understand the relationship between exposure to hazardous substances in the environment, its impact on human health, and how to protect public health. The conclusions of this column are those of the author(s) and do not necessarily represent the official position of ATSDR or CDC.

High-fat diet enhances stemness and tumorigenicity of intestinal progenitors.

Little is known about how pro-obesity diets regulate tissue stem and progenitor cell function. Here we show that high-fat diet (HFD)-induced obesity augments the numbers and function of Lgr5(+) intestinal stem cells of the mammalian intestine. Mechanistically, a HFD induces a robust peroxisome proliferator-activated receptor delta (PPAR-δ) signature in intestinal stem cells and progenitor cells (non-intestinal stem cells), and pharmacological activation of PPAR-δ recapitulates the effects of a HFD on these cells. Like a HFD, ex vivo treatment of intestinal organoid cultures with fatty acid constituents of the HFD enhances the self-renewal potential of these organoid bodies in a PPAR-δ-dependent manner. Notably, HFD- and agonist-activated PPAR-δ signalling endow organoid-initiating capacity to progenitors, and enforced PPAR-δ signalling permits these progenitors to form in vivo tumours after loss of the tumour suppressor Apc. These findings highlight how diet-modulated PPAR-δ activation alters not only the function of intestinal stem and progenitor cells, but also their capacity to initiate tumours.

Participation in and impact of a depression care management program targeting low-income minority patients in an urban community-based clinic.

BACKGROUND: African American people experience disproportionately higher rates of chronic depression, and among those affected, the condition is less likely to be detected and treated than in non-Hispanic white people. OBJECTIVE: To address this disparity in our primary care clinic, we introduced a validated framework for detecting and managing depression. METHODS: Over a 5-year period, there were 146 patients diagnosed as having depression and enrolled in a depression care management program. We evaluated the feasibility and effectiveness of that program using baseline and follow-up screening data from the Patient Health Questionnaire-9. RESULTS: The mean baseline severity score of 20.60 was reduced to 15.89 at 6 months (P < 0.001) and to 16.62 at 12 months. Patients achieved their best score, a mean of 12.93, 10.14 months after baseline (P < 0.001). The last mean severity score, after 15.47 months, was 14.60, a significant difference compared with baseline (P < 0.001). Although baseline severity scores for both groups were similar (P = 0.534), patients who remained engaged with the program demonstrated better scores and achieved greater severity score reductions from baseline to the last measure (P < 0.001). This study did not find any differences between the sexes when comparing PHQ-9 scores at baseline (P = 0.074), 6 months (P = 0.303), and 12 months (P = 0.429) and best (P = 0.875) and last (P = 0.640) scores. CONCLUSIONS: Most of the improvement was witnessed in the first 10 months of treatment. Patients with more medical comorbidities participated longer in the study than patients with fewer comorbidities. Further research could elicit the relationship between improvement in mental health and medical conditions.

Alianza terapéutica y su relación con las variables de apego del terapeuta y del consultante / Therapist and Client Attachment and its Relationship with Therapeutic Alliance

El presente estudio se diseñó con el propósito de determinar las variaciones de la alianza terapéutica (AT) a lo largo de la terapia y determinar la relación con el apego del consultante y del terapeuta. El apego se evaluó mediante la Adult Attachment Scale y la AT a través del Inventario de Alianza Terapéutica aplicado en la 1.-, la 3.- y la 10.- o última sesión. Participaron 25 terapeutas, 19 estudiantes y 6 profesionales con 1 a 4 años de experiencia clínica. Se inició con 50 consultantes mayores de edad; para la 3.- sesión quedaron 30 casos y en la última, 15. Los resultados indicaron que la seguridad del apego favorece el desarrollo de la AT evaluada por el terapeuta, en especial en la 1.- sesión. Posteriormente, la apertura a la intimidad con el consultante y la confianza en el procedimiento terapéutico benefician la AT.

This article reviews the results of a study on the development of the therapeutic alliance (TA) along the psychotherapeutic process and its relationship with therapists' and clients' attachment styles. TA was evaluated in the 1st, 3rd, and 10th or last session through the "Inventario de Alianza Terapéutica" (IAT), attachment was measured by the Adult Attachment Scale. Twenty five therapists participated, 19 senior psychology students and 6 professional psychologists with 1 to 4 years of clinical experience. The study was initiated with 50 adult people, for the 3rd session there were 30 cases, and for the last one, 15. Secure attachment of the therapist and the client associated with better TA as evaluated by the therapist. Later on, intimacy and trustfulness in the therapeutic procedures improved the TA.