Let's Get Rolling: Precise Control of Microfluidic Assay Conditions to Recapitulate Selectin-Mediated Rolling Interactions of the Leukocyte Adhesion Cascade.

The leukocyte adhesion cascade governs the recruitment of circulating immune cells from the vasculature to distal sites. The initial adhesive interactions between cell surface ligands displaying sialyl-LewisX (sLeX) and endothelial E- and P-selectins serve to slow the cells down enough to interact more closely with the surface, polarize, and exit into the tissues. Therefore, precise microfluidic assays are critical in modeling how well immune cells can interact and "roll" on selectins to slow down enough to complete further steps of the cascade. Here, we present a systematic protocol for selectin mediated rolling on recombinant surfaces and endothelial cell monolayers on polyacrylamide gels of varying stiffness. We also describe step-by-step the protocol for setting up and performing the experiment and how to analyze and present the data collected. This protocol serves to simplify and detail the procedure needed to investigate the initial selectin-mediated interactions of immune cells with the vasculature. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparing dishes for cell rolling experiments Basic Protocol 2: Fabrication of polyacrylamide gels for cell rolling experiments Alternate Protocol 1: Protein conjugation with N6 linker Alternate Protocol 2: HUVEC culturing for monolayers Basic Protocol 3: Conducting cell rolling experiments on polyacrylamide gels Basic Protocol 4: ImageJ analysis of cell rolling movies Basic Protocol 5: Quantification of Fc site density on a surface (e.g., for Fc chimeras).

Inhibition of Mac-1 allows human macrophages to migrate against the direction of shear flow on ICAM-1.

All immune cells must transit from the blood to distal sites such as the lymph nodes, bone marrow, or sites of infection. Blood borne monocytes traffic to the site of inflammation by adhering to the endothelial surface and migrating along endothelial intracellular adhesion molecule 1 (ICAM-1) by their ligand's macrophage 1 antigen (Mac-1) and lymphocyte functional antigen 1 (LFA-1) to transmigrate through the endothelium. Poor patient prognoses in chronic inflammation and tumors have been attributed to the hyper recruitment of certain types of macrophages. Therefore, targeting the binding of ICAM-1 to its respective ligands provides a novel approach to targeting the recruitment of macrophages. To that end, we determined whether the loss of Mac-1 expression could induce this upstream migration behavior by using blocking antibodies against Mac-1 to examine the effects of hydrodynamic flow on the migration of the human macrophage cell line U-937 on ICAM-1 surfaces. Blocking Mac-1 on U-937 cells led to upstream migration against the direction of shear flow on ICAM-1 surfaces. In sum, the ability of macrophages to migrate upstream when Mac-1 is blocked represents a new avenue to precisely control the differentiation, migration, and trafficking of macrophages.

Intralobar pulmonary sequestration: a rare cause of hemoptysis: a case report.

Overview and significance: Pulmonary sequestration (PS) is a rare congenital anomaly characterized by aberrant formation of nonfunctional lung tissue with anomalous systemic blood supply. Despite its rarity, PS presents significant diagnostic and management challenges, often necessitating a multidisciplinary approach for optimal patient outcomes. This case report provides insights into the clinical presentation, diagnostic modalities, and management strategies for PS. Case summary: The authors present a case of a 30-year-old male who complained of chronic cough and hemoptysis and was eventually diagnosed with intralobar PS by computed tomography (CT) imaging. The patient underwent a surgical procedure, specifically a lobectomy, to address the lung tissue. Clinical discussion: The diagnosis of intralobar PS is confirmed by CT imaging, showing features of abnormalities, including irregular cystic communication. A large area with abnormal systemic arterial supply and variable venous fluid. This patient presented with symptoms consistent with PS, including chronic cough and hemoptysis, highlighting the importance of timely diagnosis and intervention to prevent life-threatening complications. Conclusion: Lung sequestration has diagnostic challenges due to its variable clinical presentation and potential for misdiagnosis. However, advances in technology, such as CT angiography, make accurate diagnosis and precise surgical planning easier. Prompt intervention via lobectomy or transarterial embolization is important to reduce the risk of life-threatening complications associated with PS. These data highlight the importance of multidisciplinary collaboration between physicians, radiologists, and surgeons to effectively manage PS and improve patient outcomes.

Comparison of Pre- and Post-DMEK Keratometry and Total Keratometry Values for IOL Power Calculations in Eyes Undergoing Triple DMEK.

PURPOSE: To evaluate prediction accuracy of pre- and post-DMEK keratometry (K) and total keratometry (TK) values for IOL power calculations in Fuchs endothelial corneal dystrophy (FECD) eyes undergoing DMEK with cataract surgery (triple DMEK). METHODS: Retrospective cross-sectional multicenter study of 55 FECD eyes (44 patients) that underwent triple DMEK between 2019 and 2022 between two centers in USA and Europe. Swept-source optical coherence tomography biometry (IOLMaster 700) was used for pre- and post-DMEK measurements. K and TK values were used for power calculations with ten formulae (Barrett Universal II (BUII), Castrop, Cooke K6, EVO 2.0, Haigis, Hoffer Q, Hoffer QST, Holladay I, Kane, and SRK/T). Mean error, mean absolute error (MAE), standard deviation, and percentage of eyes within ±0.50/±1.00 diopters (D) were calculated. Studied formulae were additionally adjusted using a method published previously (IOLup1D Method), which increases the IOL power by 1D. While both eyes from the same patient were considered for descriptive statistics, we restricted to one eye per individual (44 eyes for statistical comparisons. RESULTS: MAEs for all formulae were lower for post-DMEK K and TK than pre-DMEK K and TK by an average of 0.24 and 0.47 D, respectively. The lowest MAE was 0.49 D for Kane using post-DMEK TK, and the highest MAE was 1.05 D for BUII using pre-DMEK TK. Most IOLup1D formulae had lower MAEs than pre-DMEK K and TK formulae. CONCLUSIONS: The IOLup1D Method should be used instead of pre-DMEK K and TK values for triple DMEK in FECD eyes. Using post-DMEK TK values for cataract surgery after DMEK provides better refractive accuracy than any of the three studied methods used for triple DMEK procedures.

Prevalence of subclinical keratoconus and impact on adults undergoing routine, uncomplicated age-related cataract extraction.

Aim: To determine the prevalence of subclinical keratoconus (SKCN) among individuals undergoing routine, uncomplicated age-related cataract surgery and its impact on visual and refractive outcomes. Patient and Methods: At a major academic ophthalmology department in the United States, we reviewed records of patients aged 50 years and older who underwent surgery from January 2011 to June 2022. We excluded patients who had poor-quality or unreliable tomographic data, previous corneal surgery, keratorefractive procedures, and significant vision-limiting ocular pathology. We defined SKCN if an eye had a Belin-Ambrósio enhanced ectasia index (BAD-D) ≥1.7, which was based on the results of a meta-analysis of large studies. In addition to the BAD-D cutoff, the eye had to deviate significantly on at least one of seven additional parameters: 1) posterior elevation at thinnest point, 2) index of vertical asymmetry, 3) index of surface variation, 4) total front higher order aberrations, 5) front vertical coma, 6) front secondary vertical coma, 7) back vertical coma. An individual had SKCN if at least one eye met the tomography-based classification and did not have manifest KCN in either eye. Visual and refractive outcomes data were acquired from patients of one experienced cataract surgeon with cases done from July 2021 to June 2022. Statistical significance was set at p < 0.05. Results: Among 5592 eyes from 3828 individuals, the prevalence of SKCN was 24.7% (95% CI, 23.4 - 26.1, 945 individuals), and the prevalence of KCN was 1.9% (95% CI, 1.6 - 2.4, 87 individuals). The prevalence of SKCN did not increase with age and was more prevalent among females and non-white races. Median post-operative month one distance-corrected visual acuity (DCVA) and proportion of eyes with improvement in DCVA were similar between normal and SKCN eyes. The proportion of eyes reaching ±0.5 and ±1.0 diopter within the refractive target were similar between normal and SKCN eyes. Conclusion: SKCN is highly prevalent and should be detected but is unlikely to have a significant deleterious effect on outcomes in routine, uncomplicated cataract surgery.

Standard vs total keratometry for intraocular lens power calculation in cataract surgery combined with DMEK.

PURPOSE: To compare the prediction accuracy of standard keratometry (K) and total keratometry (TK) for intraocular lens (IOL) power calculation in eyes undergoing combined cataract surgery and Descemet membrane endothelial keratoplasty (triple DMEK). SETTING: Tertiary care academic referral center. DESIGN: Retrospective case series. METHODS: Review of 83 eyes (63 patients) that underwent triple DMEK between 2019 and 2021. Biometry measurements were obtained using a swept-source optical biometer (IOLMaster 700). 63 eyes were used for statistical analysis. Mean error, mean absolute error (MAE), SD, median absolute error, maximum absolute error, root mean squared prediction error, and the percentage of eyes within prediction errors of ±0.50 diopters (D) and ±1.00 D were calculated for 9 multivariate and third-generation formulas using K and TK values (Barrett Universal II, Yeo EVO 2.0, Cooke K6, Kane, Pearl-DGS, Haigis, Holladay 1, Hoffer Q, and SRK/T). Formulas were additionally tested by using the prediction for an IOL power 1 D below the IOL used (IOLup1D). RESULTS: For all formulas, MAE was lower for K than for TK by an average of 0.21 D. The lowest MAE value observed was 0.67 D for "adjusted" SRK/T using K, and the highest MAE values observed were 1.24 D and 1.24 D for nonadjusted Hoffer Q and Haigis using TK, respectively. Overall, lower MAE values were observed for multivariate formulas and SRK/T. CONCLUSIONS: In triple DMEK eyes, the prediction accuracy of K was higher than that of TK. The most accurate formulas were SRK/T and multivariate formulas using K with the IOLup1D adjustment.

Choroidal thickness in non-ocular Behçet's disease - A spectral-domain OCT study.

PURPOSE: To evaluate choroidal thickness in patients with non-ocular Behçet's disease (BD) using spectral domain optical coherence tomography (SD-OCT) and to compare the results to normal eyes. METHODS: In this retrospective observational comparative study, we collected OCT and clinical data from the charts of 4 patients (7 eyes) with BD who had been referred for a screening eye exam and had a normal ocular examination. Data from 9 healthy volunteers (17 eyes) were collected as age-matched controls. The choroid was manually segmented from volume OCT scans using custom Doheny Image Reading Center OCT grading software (3D-OCTOR). Main outcome measures were choroidal thickness and intensity were compared between eyes of patients with BD and those of healthy controls. RESULTS: Eyes of patients with non-ocular BD had significantly thinner mean central subfield choroidal thickness (227.5 ± 56.93 versus 306.85 ± 17.85, P = 0.04) and central subfield choroidal volume (0.18 ± 0.04 vs 0.24 ± 0.02, P = 0.005). There was no significant difference in mean choroidal thickness in the whole ETDRS grid or in mean choroidal intensity in the central subfield and the whole ETDRS grid between eyes of patients with non-ocular BD and those of controls. CONCLUSION: This study demonstrates that BD may have subclinical manifestations in the choroid, resulting in thinning of the choroid relative to normal eyes, even without overt signs of ocular involvement.