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A 3-yr study was conducted to evaluate immune response and Cu status of yearling beef steers as a consequence of grazing tall fescue (Festuca arundinacea Schreb.) infected (E+) with the endophyte fungus Neotyphodium coenophialum ([Morgan-Jones and Gams] Glenn, Bacon, and Hanlin). During a preliminary study in 1994, 24 weanling Angus and Angus x Hereford steers were blocked by breed and weight (initial BW 271 kg; SD 25) and were randomized to E+ and low endophyte (E-) fescue in pastures at Glade Spring, VA. Grazing began in April and was discontinued in July. In 1995 and 1996, 24 weanling Angus and Angus x Hereford steers (initial BW 249 kg, SD 20 and 240 kg, SD 15, respectively) were randomized to the E+ and E- pastures at Glade Spring during each year. Grazing began in April and continued until September in 1995 and October in 1996. In 1994, steers that grazed E+ fescue exhibited lower (P < .05) phagocytic activity, major histocompatibility complex (MHC) class II expression, ceruloplasmin, and serum Cu than steers that grazed E- tall fescue. During 1995, steers grazing E+ fescue had lower (P < .05) phagocytic activity and MHC class II expression than steers that grazed E- fescue. In 1996, one-half of the steers within each paddock received a Cu oxide bolus at the beginning of the grazing season. During 1996, phagocytic activity was lower (P < .01) and MHC class II expression tended (P < .07) to be lower in steers that grazed E+ tall fescue than in steers that grazed E- tall fescue. Copper supplementation increased (P < .05) MHC class II expression in July regardless of endophyte status over nonsupplemented steers. Steers that grazed E- tall fescue had higher (P < .05) plasma or serum Cu concentrations than steers that grazed E+ tall fescue in each year of the study. These data indicate that the endophyte compromised the immune function of grazing steers, and the data suggest a relationship with depressed Cu status. Key Words: Immune Response, Steers, Endophytes, Copper, Forage, Minerals

Although the early identification of patients with suboptimal nutritional status can allow the implementation of nutritional intervention to enhance the ability of the body to fight infection and disease, currently no definitive test of nutritional status exists. Therefore, this study was conducted to identify possible functional indicators of acute nutritional deprivation. The effects of total nutritional deprivation and subsequent refeeding on lymphocyte functions and subpopulations were examined in 23 healthy cats. Peripheral blood samples were analyzed at various times during food deprivation and refeeding periods. During the food deprivation period, decreases were observed in leukocyte number (P: < 0.05), lymphocyte number (P: < 0.05), percentage of CD4(+) cells [before stimulation with concanavalin-A (Con-A); P: < 0.05] and the CD4/CD8 ratio (before stimulation with Con-A; P: < 0.01) compared with d 0. Increases were observed in the percentage of CD8(+) cells [before (P: < 0.05) and after (P: < 0.01) stimulation with Con-A] and in intracellular calcium (P: < 0.01) during acute starvation. During the refeeding period, increases were observed in the percentage of CD4(+) cells (before and after stimulation with Con-A; P: < 0.01), the percentage of CD8(+) cells (before stimulation with Con-A; P: < 0.05) and lymphocyte number (P: < 0.05) compared with d 7. Lymphocyte proliferative capacity tended to decrease (P: = 0.07) during starvation and increased (P: < 0.01) during the refeeding period. These findings suggest that a 7-d starvation period had immunosuppressive effects on cats and that these effects were not completely normalized during 7 d of refeeding. CD4(+)/CD8(+) subset alterations and CD4/CD8 ratio in conjunction with lymphocyte proliferation may be useful as indices of nutritional status. [ABSTRACT FROM AUTHOR]

Consuming diets enriched with either (n-6) or (n-3) fatty acids (FA) will subsequently influence the proportion of cell membrane–associated lipids, arachidonic acid (AA) and eicosapentaenoic acid (EPA). Recent investigations suggest that the mechanism underlying the effectiveness of (n-3) FA supplementation involves the reduction of inflammatory mediators (Vaughn et al. 1994). In addition to reducing levels of inflammatory mediators, (n-3) FA have been associated with altered platelet function. Activation of platelets with high concentrations of (n-6) or (n-3) FA in the cell membrane results in the release of increased amounts of proaggregatory thromboxane A2 (TXA2) or antiaggregatory thromboxane A3 (TXA3) metabolites (Hall 1996), respectively. Studies regarding the possible detrimental effects that may occur from intake of dietary (n-3) FA have yielded conflicting results regarding platelet function (Casali et al. 1986, Kristensen et al. 1989, Landhmore et al. 1986). Currently, pet foods are being formulated to maximize the anti-inflammatory effect of (n-3) FA. Although the benefits of increasing dietary (n-3) FA have been studied, potential adverse effects of feeding diets enriched in (n-3) FA on a long-term basis have not been evaluated. Therefore, the objective of this study was to determine the effects of altering dietary (n-6) to (n-3) FA ratios on platelet aggregation, platelet activation, blood coagulation indices and bleeding time in cats. Materials and methods. Animals and facilities. Twelve domestic short-haired cats, six neutered males and six spayed females, (average age 4 y) were used in this experiment. Cats were housed and fed in individual metal cages in a climate-controlled environment at the Virginia-Maryland Regional College of Veterinary Medicine for the duration of the study. All cats were fed a nutritionally complete dry diet to maintain an optimal body weight and condition score before the start of the study. The prestudy diet contained a (n-6):(n-3) fatty acid ratio of 12:1. The experimental protocol was reviewed and approved by the Virginia Tech Animal Care and Use Committee according to NIH guidelines (NRC 1985). Experimental protocol. Cats were allotted by gender and weight to dietary treatments as follows: enriched (n-6), [(n-6):(n-3) FA ratio 25:1]; control, (12:1); or enriched (n-3), (1.3:1). Experimental diets were formulated by Hill’s Science and Technology Center, Topeka, KS. Fatty acid ratios were manipulated by incorporating specific FA sources into each of the three treatment diets (corn oil, choice white grease or menhaden oil, respectively). During the 16-wk study period, whole blood was collected via jugular venipuncture on d 0, 56 and 112 for evaluation of platelet function, coagulation parameters and fibrinogen concentration. Toenail bleeding time was recorded on d 0 and 112. Platelet isolation. Whole blood was collected via jugular venipuncture into syringes containing 3.8% trisodium citrate (9 parts blood:1 part anticoagulant), and platelet-rich plasma (PRP) was harvested according to the procedure described by Welles et al. (1994). Platelet counts were determined by using a Baker System 9000 Hematology Series Cell Counter (Allentown, PA). Samples of PRP were diluted to 300,000 platelets/ µL with autologous platelet-poor plasma (PPP). Platelet aggregation. Platelet aggregation was performed on diluted PRP stimulated with 50 µmol/L ADP (grade I: Sigma Chemical, St. Louis, MO) using a dual-channel aggregometer (Payton Series 300BD-5, Buffalo, NY). The procedure has been previously described (Welles et al. 1994). Platelet activation. Platelet activation was measured using a modified technique previously reported by Welles et al. (1994). Briefly, PRP stimulated with 50 µmol/L ADP was labeled with fluorescein- conjugated goat F[Ab] monoclonal antibody (Cappel-Organon Teknika, Durham, NC). Fluorescence was measured on the platelets bound with fluorescein-labeled antifibrinogen using a Coulter Epics XL-MCL Flow Cytometer. Determination of bleeding time, coagulation indices, and fibrinogen. Cats were sedated with a combination of acepromazine (0.11 mg/kg, intramuscular) and ketamine HCl (22 mg/kg, intramuscular); one nail was trimmed 2 mm into the capillary bed and gently blotted every 10 s until a clot formed. Time to clot formation was recorded. Whole blood was collected via jugular venipuncture for determination of activated partial thromboplastin time (APTT), one-stage prothrombin time (OSPT) and fibrinogen concentration with the use of a Sysmex Coagulation Analyzer 1000. Statistical analysis. Data were tested for normality and homogeneity of variance. An ANOVA (SAS/STAT Version 5.0, SAS Institute, Cary, NC) was used to determine dietary treatment effects on platelet count, platelet aggregation, platelet activation, bleeding time, fibrinogen concentration, APTT and OSPT. Tukey’s studentized range test (HSD) was used for means comparisons. The General Linear Models procedure for repeated-measures ANOVA was used to determine within-subject effects and compare response changes over time. Data were considered significant at P < 0.05. Results. Diet analyses are presented in Table 1. Cats fed the enriched (n-3) FA diet for 112 d exhibited an increased (P < 0.05) toenail bleeding time compared with cats fed either the enriched (n-6) FA or control diet (6.1 vs. 3.2 and 3.2 min, respectively). Peak aggregation of platelets from cats fed the enriched (n-3) FA diet was lower (P < 0.05) compared with both the enriched (n-6) FA and control diet on d 112 (Fig. 1). Platelet activation (Fig. 2) tended (P = 0.15) to be higher in the enriched (n-6) FA and control diet groups on d 56 and 112 compared with the enriched (n-3) FA diet group. There was no difference between treatment groups for OSPT, APTT and fibrinogen concentration throughout the study period (data not shown). Discussion. Manipulation of dietary (n-6):(n-3) ratios does not appear to affect coagulation indices such as APTT and OSPT or platelet count in human studies, but platelet function does appear to be altered (Kristensen et al. 1989). The effects of FA supplementation for 4 or 6 wk on bleeding time in dogs have been contradictory (Casali et al. 1986, Landhmore et al. 1986). In this study, bleeding time was significantly increased in cats fed an enriched (n-3) FA diet for 16 wk. Indices of the intrinsic, extrinsic and common coagulation pathways (APTT and OSPT) were not different among cats in the dietary treatment groups throughout the study. Therefore, it is unlikely that alterations in bleeding time were a function of abnormalities in the coagulation cascade. Alterations in bleeding time due to increased dietary (n-3) FA levels may occur in less time than 16 wk; however, that information could not be determined from our study design. The ability of activated platelets to aggregate appeared to be influenced by the ratio of (n-6) to (n-3) FA in the diet as shown in Figure 1. As the level of dietary (n-6) FA decreased, there would be proportionately less AA incorporated into platelet membranes and, therefore, less AA available for conversion to the proaggregatory TXA2. As the concentrations of TXA2 decreased, the ability of platelets to aggregate would diminish. Kristensen et al. (1989) showed a similar effect on human platelet aggregation when fish-based diets were fed. Aggregation of platelets is dependent on or enhanced by fibrinogen binding to platelets. Alteration of platelet membrane glycoprotein complexes, which form a functional receptor for fibrinogen, is an activation- dependent platelet surface change (Welles et al. 1994). The method of measuring platelet activation in this study used an antifibrinogen antibody, which requires the presence of adequate fibrinogen to first bind to the exposed fibrinogen receptors of activated platelets. Our results indicated that platelets derived from cats fed the enriched (n-3) FA diet tended to exhibit decreased activation compared with platelets from cats fed the enriched (n-6) or control diet; fibrinogen concentration did not differ among treatment groups during the study, and values were within the normal range for cats (Duncan and Prasse 1986). This suggested that a decrease in platelet activation resulted in a decrease in aggregatory ability and that this sequence of events was not limited by fibrinogen concentration. Saynor and Gillott (1992) reported that intake by human subjects of diets containing high concentrations of fish oil for over a 7-y period significantly reduced fibrinogen. Possible longer-term effects on fibrinogen concentration of felines fed (n-3) FA-enriched diets may warrant further study with a larger sample size. The results from this study suggest that prolonged (112 d) intake of enriched (n-3) FA diets in cats alters platelet aggregation and bleeding time, such that there were detrimental effects on platelet function. Specific medical conditions in which an enriched (n-3) FA diet or FA supplementation may be indicated would require long-term feeding to confirm the viability of a desired effect. [ABSTRACT FROM AUTHOR]

Outer retinal degenerations, including age-related macular degeneration (AMD), are characterized by photoreceptor and retinal pigment epithelium (RPE) atrophy. In these blinding diseases, macrophages accumulate at atrophic sites, but their ontogeny and niche specialization remain poorly understood, especially in humans. We uncovered a unique profile of microglia, marked by galectin-3 upregulation, at atrophic sites in mouse models of retinal degeneration and human AMD. In disease models, conditional deletion of galectin-3 in microglia led to phagocytosis defects and consequent augmented photoreceptor death, RPE damage, and vision loss, indicating protective roles. Mechanistically, Trem2 signaling orchestrated microglial migration to atrophic sites and induced galectin-3 expression. Moreover, pharmacologic Trem2 agonization led to heightened protection but in a galectin-3-dependent manner. In elderly human subjects, we identified this highly conserved microglial population that expressed galectin-3 and Trem2. This population was significantly enriched in the macular RPE-choroid of AMD subjects. Collectively, our findings reveal a neuroprotective population of microglia and a potential therapeutic target for mitigating retinal degeneration., (© 2024 Yu et al.)

Degenerative diseases of the outer retina, including age-related macular degeneration (AMD), are characterized by atrophy of photoreceptors and retinal pigment epithelium (RPE). In these blinding diseases, macrophages are known to accumulate ectopically at sites of atrophy, but their ontogeny and functional specialization within this atrophic niche remain poorly understood, especially in the human context. Here, we uncovered a transcriptionally unique profile of microglia, marked by galectin-3 upregulation, at atrophic sites in mouse models of retinal degeneration and in human AMD. Using disease models, we found that conditional deletion of galectin-3 in microglia led to defects in phagocytosis and consequent augmented photoreceptor death, RPE damage and vision loss, suggestive of a protective role. Mechanistically, Trem2 signaling orchestrated the migration of microglial cells to sites of atrophy, and there, induced galectin-3 expression. Moreover, pharmacologic Trem2 agonization led to heightened protection, but only in a galectin-3-dependent manner, further signifying the functional interdependence of these two molecules. Likewise in elderly human subjects, we identified a highly conserved population of microglia at the transcriptomic, protein and spatial levels, and this population was enriched in the macular region of postmortem AMD subjects. Collectively, our findings reveal an atrophy-associated specialization of microglia that restricts the progression of retinal degeneration in mice and further suggest that these protective microglia are conserved in AMD.

Background: Sexual dimorphisms in immune responses contribute to coronavirus disease 2019 (COVID-19) outcomes, but the mechanisms governing this disparity remain incompletely understood., Methods: We carried out sex-balanced sampling of peripheral blood mononuclear cells from hospitalized and non-hospitalized individuals with confirmed COVID-19, uninfected close contacts, and healthy control individuals for 36-color flow cytometry and single-cell RNA sequencing., Findings: Our results revealed a pronounced reduction of circulating mucosal-associated invariant T (MAIT) cells in infected females. Integration of published COVID-19 airway tissue datasets suggests that this reduction represented a major wave of MAIT cell extravasation during early infection in females. Moreover, MAIT cells from females possessed an immunologically active gene signature, whereas cells from males were pro-apoptotic., Conclusions: Our findings uncover a female-specific protective MAIT cell profile, potentially shedding light on reduced COVID-19 susceptibility in females., Funding: This work was supported by NIH/NIAID (U01AI066569 and UM1AI104681), the Defense Advanced Projects Agency (DARPA; N66001-09-C-2082 and HR0011-17-2-0069), the Veterans Affairs Health System, and Virology Quality Assurance (VQA; 75N93019C00015). The content is solely the responsibility of the authors and does not necessarily represent the official view of the National Institutes of Health. COVID-19 samples were processed under Biosafety level 2 (BSL-2) with aerosol management enhancement or BSL-3 in the Duke Regional Biocontainment Laboratory, which received partial support for construction from NIH/NIAID (UC6AI058607)., Competing Interests: M.T.M. reports grants for biomarker diagnostics from the Defense Advanced Research Projects Agency (DARPA), National Institutes of Health (NIH), Sanofi, and the Department of Veterans Affairs. T.W.B. reports grants from DARPA and is a consultant for Predigen. M.T.M., T.W.B., E.L.T., and C.W.W. report pending patents on molecular methods to diagnose and treat respiratory infections. E.L.T. reports grants on biomarker diagnostics from DARPA and the NIH/Antibacterial Resistance Leadership Group (ARLG) and an ownership stake in Predigen. G.S.G. reports an ownership stake in Predigen. C.W.W. reports grants for biomarker diagnostics from DARPA, NIH/ARLG, Predigen, and Sanofi and has received consultancy fees from bioMerieux, Roche, Biofire, Giner, and Biomeme., (© 2021 Elsevier Inc.)

Elevated intraocular pressure (IOP) narrows Schlemm's canal (SC), theoretically increasing luminal shear stress. Using engineered adenoviruses containing a functional fragment of the shear-responsive endothelial nitric oxide synthase (eNOS) promoter, we tested effects of shear stress and elevated flow rate on reporter expression in vitro and ex vivo. Cultured human umbilical vein endothelial cells (HUVECs) and SC cells were transduced with adenovirus containing eNOS promoter driving secreted alkaline phosphatase (SEAP) or green fluorescent protein (GFP) and subjected to shear stress. In parallel, human anterior segments were perfused under controlled flow. After delivering adenoviruses to the SC lumen by retroperfusion, the flow rate in one anterior segment of pair was increased to double pressure. In response to high shear stress, HUVECs and SC cells expressed more SEAP and GFP than control. Similarly, human anterior segments perfused at higher flow rates released significantly more nitrites and SEAP into perfusion effluent, and SC cells expressed increased GFP near collector channel ostia compared to control. These data establish that engineered adenoviruses have the capacity to quantify and localize shear stress experienced by endothelial cells. This is the first in situ demonstration of shear-mediated SC mechanobiology as a key IOP-sensing mechanism necessary for IOP homeostasis.

Progressive rod-cone degeneration (PRCD) is a small protein residing in the light-sensitive disc membranes of the photoreceptor outer segment. Until now, the function of PRCD has remained enigmatic despite multiple demonstrations that its mutations cause blindness in humans and dogs. Here, we generated a PRCD knockout mouse and observed a striking defect in disc morphogenesis, whereby newly forming discs do not properly flatten. This leads to the budding of disc-derived vesicles, specifically at the site of disc morphogenesis, which accumulate in the interphotoreceptor matrix. The defect in nascent disc flattening only minimally alters the photoreceptor outer segment architecture beyond the site of new disc formation and does not affect the abundance of outer segment proteins and the photoreceptor's ability to generate responses to light. Interestingly, the retinal pigment epithelium, responsible for normal phagocytosis of shed outer segment material, lacks the capacity to clear the disc-derived vesicles. This deficiency is partially compensated by a unique pattern of microglial migration to the site of disc formation where they actively phagocytize vesicles. However, the microglial response is insufficient to prevent vesicular accumulation and photoreceptors of PRCD knockout mice undergo slow, progressive degeneration. Taken together, these data show that the function of PRCD is to keep evaginating membranes of new discs tightly apposed to each other, which is essential for the high fidelity of photoreceptor disc morphogenesis and photoreceptor survival., Competing Interests: The authors declare no conflict of interest.

Microglia from different nervous system regions are molecularly and anatomically distinct, but whether they also have different functions is unknown. We combined lineage tracing, single-cell transcriptomics, and electrophysiology of the mouse retina and showed that adult retinal microglia shared a common developmental lineage and were long-lived but resided in two distinct niches. Microglia in these niches differed in their interleukin-34 dependency and functional contribution to visual-information processing. During certain retinal-degeneration models, microglia from both pools relocated to the subretinal space, an inducible disease-associated niche that was poorly accessible to monocyte-derived cells. This microglial transition involved transcriptional reprogramming of microglia, characterized by reduced expression of homeostatic checkpoint genes and upregulation of injury-responsive genes. This transition was associated with protection of the retinal pigmented epithelium from damage caused by disease. Together, our data demonstrate that microglial function varies by retinal niche, thereby shedding light on the significance of microglia heterogeneity., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Meibomian glands (MGs) are sebaceous glands of the eyelid margin that secrete lipids needed to avert tear evaporation and to help maintain ocular surface homeostasis. Obstruction of MGs or other forms of MG dysfunction can promote chronic diseases of the ocular surface. Although chronic eyelid inflammation, such as allergic eye disease, is an associated risk factor for obstructive MG dysfunction, it is not clear whether inflammatory processes contribute to the pathophysiology of MG obstruction. We show that polymorphonuclear neutrophils (PMNs) promoted MG obstruction in a chronic inflammatory model of allergic eye disease in mice. Analysis of leukocytes in tears of patients with MG dysfunction showed an increase in PMN numbers compared to healthy subjects. Moreover, PMN numbers in tears positively correlated with clinical severity of MG dysfunction. Our findings point to a role for PMNs in the pathogenesis and progression of MG dysfunction., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Objectives: To compare results of a nonradioactive colorimetric microplate assay with results of a traditional radioactive proliferation assay for determination of its use as a reliable and accurate alternative method for determination of proliferative activity of feline lymphocytes., Sample Population: Blood samples from 10 clinically normal domestic shorthair cats., Procedure: Double-density gradient separation was used to isolate mononuclear cells. Isolated cells were stimulated with various concentrations of concanavalin A (Con-A) and cultured for 72 hours. Lymphocyte proliferation was measured by radioactive ([3H]thymidine) and nonradioactive (colorimetric) techniques. Immunophenotypic analysis with feline-specific CD4+ and CD8+ monoclonal antibody was performed, using flow cytometry., Results: Mononuclear cells were successfully isolated (97 to 99% purity and viability) from blood samples. A similar dose-dependent proliferative response to Con-A stimulation was measured with [3H]thymidine incorporation and the colorimetric assay. For both techniques, concentrations of 0.1 and 1.0 microg of Con-A/ml were submitogenic, and 100 microg/ml was toxic to cultured cells. For both techniques, maximal proliferation was observed with 5 microg of Con-A/ml., Conclusions and Clinical Relevance: These results indicate that the nonradioactive colorimetric technique is a reliable and accurate method for measuring proliferative activity of feline lymphocytes. Clinically, this assay can be used as part of a screening process to determine immunocompetence of at-risk cats and to evaluate treatments for cats with immune-mediated or T-cell-dependent diseases.