Immune Cell Counts Research Articles

Effect of supplementation with algae β-glucans on performance, health, and blood metabolites of Holstein dairy calves.

Studies have shown that β-glucans extracted from the cell wall of cereals, algae, and yeasts have been associated with improved immune function. However, it is unknown whether algae β-glucan supplementation affects the performance, blood metabolites, or cell counts of immune cells in dairy calves. The objective of this randomized clinical trial was to evaluate whether supplementation of β-glucans to milk replacer in dairy calves fed 6 L/d improved growth performance and fecal status and altered the blood metabolite profile. In this trial, we enrolled Holstein calves (n = 34) at birth (body weight 36.38 ± 1.33 kg; mean ± standard deviation) to receive, from 1 d of age, either 2 g/d algae β-glucans mixed into 6 L/d of milk replacer (22.4% crude protein and 16.2% fat) or an unsupplemented milk replacer (control). The calves were blocked in pairs according to birth weight, sex, and date of birth (up to 5 d difference). Calves were housed individually, and calf starter (24.7% crude protein and 13.9% neutral detergent fiber) was offered ad libitum based on orts of the previous day until 56 d of age (end of the trial). Body weight was measured weekly, and health checks and daily fecal consistency were evaluated daily in every calf by the same observer. Calves with 2 consecutive days of loose feces that sifted through bedding were considered diarrhea positive. We used a linear mixed effects model to evaluate the effects of β-glucan supplementation fed during the preweaning period on performance (average daily gain), final weight, feed efficiency (FE), white blood cell count, and selected blood metabolites, repeated by time. A generalized linear mixed effects model was also run to evaluate the likelihood of a diarrhea bout in the first 28 d of life, controlling for the calf as the subject with a logistic distribution. We included age, serum total protein at 48 h, and birth weight as covariates. At 56 d, β-glucan-supplemented calves weighed more than control calves (56.3 vs. 51.5 kg). Treatment had no effect on total starter intake, but there was a treatment by age interaction for FE, with greater FE for β-glucan-supplemented calves in wk 3 and 5 of age. There was only a tendency for average daily gain to be greater in supplemented calves than in control calves for the duration of the study. Furthermore, control calves had 14.66 [95% confidence interval (95% CI): 9.87-21.77] times greater odds of having a diarrheal bout than β-glucan-supplemented calves. Control calves had 12.70 (95% CI: 8.82-18.28) times greater odds of having an additional day with an abnormal fecal score compared with β-glucan-supplemented calves, suggesting that supplementation ameliorated diarrhea severity. We found no association of treatment with concentrations of serum total protein, albumin, creatinine, or glucose during the preweaning period. Our findings suggest that dietary supplementation of 2 g/d of algae β-glucans to milk replacer improved fecal status and may affect growth, as evidenced by a higher weaning weight, compared with control calves. Future studies should explore the effect of algae β-glucans on lower-gut physiology and digestibility in dairy calves.

Stocking density, restricted trough space, and implications for sheep behaviour and biological functioning.

Stocking density and trough space allowance can potentially impact sheep welfare during live export voyages. The aim of this study was to assess the welfare implications for sheep housed at five allometric stocking densities, with either unrestricted or restricted trough space allowance. Merino wethers (n = 720) were housed in 40 pens of 18 heads for 18 days. Two 5-min continuous focal animal observations (n = 3/pen) were conducted on days 3, 5, 11, and 17. Scan sampling of standing and lying behaviours were conducted on the same days at hourly intervals. Live weights and immune cell counts were quantified at the start and end of the experiment, as well as faecal glucocorticoid metabolites (FGCMs), which were also assessed on days 6 and 12. Focal animals housed at higher stocking densities spent less time lying during one of the continuous observation periods, but no important effects on the overall number of animals lying or on the synchronicity of lying were evident. The scan sampling results indicated that the expression of some preferred lying positions was impaired at high stocking densities, and that high stocking densities also resulted in increased agonistic social interactions and displacement events at the start of the trial. There was a slight reduction in day 18 live weights for animals housed at higher stocking densities, but FGCM concentrations and immune cell counts were essentially unaffected. Trough space had no important effects on day 18 live weight, FGCM concentrations, or immune cell counts, and had limited effects on sheep behaviour. The lack of important impacts on biological fitness traits suggests that the behavioural responses observed were sufficient in allowing sheep to cope with their environment. However, we provide evidence that the provision of additional space is beneficial in reducing the time it takes for animals to adapt to their environment and to facilitate the expression of some preferred lying positions. While designed to emulate certain conditions relevant during live export voyages, some factors that may induce stress during this mode of transport were not present such as heat and ocean swell, so the conclusions must be interpreted in the context of the experimental conditions.

Immunomodulatory roles of propofol and sevoflurane in murine models of breast cancer

Background Anesthetics are emerging regulators of cancer progression. Here we aim to explore the immunomodulatory roles of two common anesthetics, propofol and sevoflurane in breast cancer progression. Methods On murine 4T1 breast cancer models, we isolated immune cells from peripheral blood after treatment with propofol and sevoflurane during tumor resection. The CD3, CD4, and CD8 expression of these immune cells were compared using flow cytometry to determine which immune cells were prominently affected by propofol and sevoflurane. Serum cytokine levels were determined using enzyme-linked immunosorbent assay (ELISA). Metastases in lung and liver tissues were counted. In MDA-MB-231 tumor models, the cell count of immune cells was determined. The cytotoxicity of T cells and natural killing cells in co-culture after propofol and sevoflurane treatment were determined using the LDH assay. Results In the 4T1 breast cancer model, T-lymphocytes showed significant cell count reduction. TNF-α was significantly upregulated at 3 and 24 h after treatment, while IL-2 and IFN-γ showed transient upregulation at 3 h after treatment. Propofol and sevoflurane increased the number of metastases in the lung and liver after primary tumor resection. In the MDA-MB-231 tumor model, CD3 and CD4 cells were also prominently reduced by propofol and sevoflurane treatment. In vitro, the proliferation and cell-killing activity of T cells and NK cells were also attenuated. Conclusions Propofol and sevoflurane had significant effects in modulating cancer progression through their immunosuppressive role. The proliferation and killing activity of anti-tumor immune cells can be suppressed by propofol and sevoflurane.

315.5: Mesenchymal Stromal Cell Therapy as Promising Option To Increase Organ Availability and Reduce Graft Failure

Introduction: Lung transplantation (LTx) is an established therapeutic option for end-stage pulmonary disease. However, waiting lists are long and the treatments remains hampered by donor lung scarcity accompanied with high early and late mortality rates. Novel treatment options to regenerate marginal donor lungs turned down for transplant are needed to combat both waiting list mortality and post-operative complications such as primary graft dysfunction (PGD). Although donor numbers increase, donor organ utility is low due to acute lung injury (ALI) which can be induced by gastric aspiration. Mesenchymal stromal cells (MSCs) have caught attention for organ regeneration as they display an array of potentially beneficial effects: they are known to be immunomodulatory, antifibrotic and microbiocidal. MSCs have demonstrated tissue regenerative capacities as documented in preclinical studies, e.g., improved function of diseased lungs. Aim: The aim of the current study is to evaluate the effect of MSCs administered to aspiration damaged lungs during ex vivo lung perfusion (EVLP) and to recipients post transplantation with a focus on their capacity to improve damaged lungs and to decrease the incidence of PGD at 72h post transplantation. Materials and Methods: Lung injury was induced in 12 donor pigs using gastric content with a pH of 2. The donor-recipient pairs were randomized to receive MSCs or placebo treatment during the beginning of EVLP and post LTx. Lungs were treated with EVLP for 4 hours followed by a left lung transplantation. The recipient was kept under anesthesia and closely monitored for three days post LTx. In the final phase of the experimental setup an isolated assessment of the transplanted lung was achieved following a right pneumonectomy. Treatment effects were assessed by clinically relevant parameters, including PaO2/FiO2 ratio, pulmonary vascular resistance (PVR), and lactate, further by histopathological changes, immune cell counts in peripheral blood and cytokine and chemokine levels in plasma and bronchoalveolar lavage fluid during the course of the experiment. Results: The lung functionality as evaluated by the PaO2/FiO2 ratios showed significantly increased lung function in treated recipients compared to both the non-treated recipients and to all lungs at the time of confirmed ALI. A tendency of higher PVR was seen among the non-treated recipients however, not significantly. Lactate was unchanged in both groups. Lung injury scores were also reduced in the treated group as compared to the non-treated group and to the lungs after lung injury was induced. On the third day of follow-up of the recipients, all recipients in the treatment group had PGD grade 0, while the non-treated group consisted of one recipient with grade 0, three with grade 2 and two with grade 3. Conclusion: MSC treatment emerges as a promising therapeutic option for restoring injured donor lungs for reacceptance into the donor pool and decreasing the incidence of PGD.

Serum cytokine levels are associated with tumor progression during FOLFIRINOX chemotherapy and overall survival in pancreatic cancer patients.

BackgroundBiomarkers predicting treatment response may be used to stratify patients with pancreatic ductal adenocarcinoma (PDAC) for available therapies. The aim of this study was to evaluate the association of circulating cytokines with FOLFIRINOX response and with overall survival (OS).MethodsSerum samples were collected before start and after the first cycle of FOLFIRINOX from patients with PDAC (n=83) of all disease stages. Overall, 34 circulating cytokines were analyzed with a multiplex immunoassay. In addition, changes in peripheral blood immune cell counts were determined by flow cytometry to correlate with differences in cytokine levels. Chemotherapy response was determined by CT scans with the RECIST 1.1 criteria, as disease control (n=64) or progressive disease (n=19) within eight cycles of FOLFIRINOX.ResultsPatients with high serum IL-1RA concentrations after one cycle of chemotherapy were less likely to have tumor progression during FOLFIRINOX (OR 0.25, P=0.040). Increase of circulating IL-1RA concentrations correlated with increase of total, classical (CD14+CD16-), and non-classical monocytes (CD14-CD16+), and dendritic cells. In multivariable cox regression, including the variables chemotherapy response outcome and baseline CA19-9 level, serum concentrations of IL-7 (HR 2.14, P=0.010), IL-18 (HR 2.00, P=0.020), and MIP-1β (HR 0.51, P=0.025) after one cycle of FOLFIRINOX showed correlations with OS.ConclusionsCirculating IL-1RA, IL-7, IL-18, and MIP-1β concentrations are biomarkers associated with FOLFIRINOX response in PDAC patients, suggesting an important role for specific immune cells in chemotherapy response and PDAC progression. Cytokine-based treatment might improve patient outcome and should be evaluated in future studies.

Serologic response and safety of COVID-19 vaccination in HSCT or CAR T-cell recipients: a systematic review and meta-analysis

BackgroundPatients receiving hematopoietic stem cell transplantation (HSCT) or chimeric antigen receptor T cell (CAR T-cell) therapy are immunocompromised and at high risk of viral infection, including SAR2-CoV-2 infection. However, the effectiveness and safety of COVID-19 vaccines in these recipients is not well characterized. The present meta-analysis evaluated the serologic response and safety of COVID-19 vaccines in these population.MethodsLiterature databases (MEDLINE, EMBASE, Web of Science, MedRvix and BioRvix) were searched for original studies with serologic response post COVID-19 vaccination in HSCT or CAR T-cell recipients published until July 14, 2022. The analysis included 27 observational studies with a total of 2899 patients receiving allogeneic HSCT (2506), autologous HSCT (286) or CAR T-cell therapy (107), and 683 healthy participants with serologic response data. Random effects models were used to pool the rate of serologic response to COVID-19 vaccination in HSCT or CAR T-cell recipients and odds ratio comparing with healthy controls.ResultsThe pooled seropositivity rates in HSCT and CAR T-cell recipients were 0.624 [0.506–0.729] for one dose, 0.745 [0.712–0.776] for two doses. The rates were significantly lower than those in healthy controls (nearly 100%). In subgroup analysis, CAR T-cell recipients exhibited an even lower seroconversion rate (one dose: 0.204 [0.094–0.386]; two doses: 0.277 [0.190–0.386]) than HSCT counterparts (one dose: 0.779 [0.666–0.862]; two doses: 0.793 [0.762–0.821]). The rates were comparable between autologous and allogeneic HSCT recipients. Other possible impact factors related to seropositivity were time interval between therapy and vaccination, use of immunosuppressive drugs and immune cell counts. Most vaccine-related adverse effects were mild and resolvable, comparable to general population.ConclusionsThis analysis revealed a diminished response to COVID-19 vaccines in HSCT or CAR T-cell recipients. Our findings may inform regular COVID-19 vaccination at appropriate intervals after HSCT or CAR T-cell therapy.

Impact of persistent antigenic challenges and splenectomy on immune cells in β-Thalassemic patients.

Infection is common in thalassemia patients and is one of the leading causes of death. It's still unclear why these individuals are so sensitive to infection. There is strong evidence that a deficiency in the functioning of phagocytic cells plays a key role in the weakened resistance to pathogenic bacteria. The purpose of this study was to investigate the function of phagocytic cells by comparing the serum levels of granulocyte macrophage-colony stimulating factor (GM-CSF) and Neopterin in thalassemia patients to healthy people. The study included 50 thalassemia patients and 30 healthy controls. Enzyme-linked immunosorbent assay (ELISA) was applied to estimate the serum levels of GM-CSF and Neopterin. Serum levels of GM-CSF were significantly elevated in thalassemia patients when compared to healthy people (p < 0.05), while serum levels of Neopterin showed no significant change between thalassemia patients and healthy controls. Both serum levels of GM-CSF and Neopterin showed no significant difference between Splenectomized and healthy controls. Total leukocyte counts, lymphocytes, MID (monocytes), platelets, and RBC were all significantly higher in thalassemia patients compared to healthy controls. But, granulocyte counts showed no significant difference between the thalassemia patients and the healthy controls. On the other hand, total leukocytes, monocytes, lymphocytes and platelets counts were significantly raised in splenectomized patients when compared to healthy controls and non-splenectomized patients, respectively. We came to the conclusion that thalassemia patients have a high immune cell count, which is most likely due to the antigenic difficulties posed by blood transfusions. On the other hand, these patients have an impaired immune system.

Seeking and identifying time window of antibiotic treatment under in vivo guidance of PbS QDs clustered microspheres based NIR-II fluorescence imaging

During bacterial infection, it is crucial to duly implement antibiotic treatment at a bottleneck time window when uncontrollable bacteria invasion exacerbates the condition. Here, we present a second near-infrared (NIR-II) fluorescence imaging strategy based on lead sulfide quantum dots clustered microspheres (PbS QDs@microspheres) to guide seeking and identifying the time window of antibiotic treatment. Our PbS QDs@microspheres with an average size of 510 nm were prepared through cross-linking process, demonstrating nine times higher photoluminescence (PL) intensity than the original PbS QDs, along with deep optical tissue penetration in muscle and 50% fat + 50% muscle. Notably, monitoring of bacterial infection in a mouse model of joint infection was successfully achieved by utilizing PbS QDs@microspheres to label staphylococcus aureus (S. aureus) in vivo, shown by a wave pattern of PL intensity lasting from 1 h to 3 d post-infection. Instructed by the NIR-II fluorescence signals from the intra-articularly injected S. aureus, 1 h to 3 d post-infection was sought to be a period of bacteria burst, along with relatively low immune cells count (neutrophils, B lymphocytes and T lymphocytes). On this basis, four therapeutic regimens (antibiotic administration at 1 h, 2 h, 1 d and 3 d post-infection) of antibiotic treatment were implemented to identify the time window, among which the 3 d group resulted in a survival rate of 50%, significantly lower than the other three groups (P = 0.028). Both the results of blood culture and histopathological analysis denoted that antibiotic administration at 3 d post-infection induced higher inflammatory reaction during the recovery period. In short, under the in vivo guidance of NIR-II fluorescence imaging based on our successfully prepared PbS QDs@microspheres, 1 h to 3 d post-infection was eventually identified as the time window of antibiotictreatment for joint infection of S. aureus in a mouse model. Thus, this study would further assist in deciphering precise therapeutic regimens during antibiotic treatment.

Infection dynamics of Salmonella Infantis vary considerably between chicken lines

ABSTRACT Salmonella (S.) Infantis is the most common serovar in broilers and broiler meat in the European Union. In the field, fast-growing broilers are reported to be more affected than slow-growing and layer birds. The present study investigated the infection dynamics and immunological response of four chicken lines in the course of a S. Infantis infection. Two commercial chicken lines, Ross 308 and Hubbard ISA-JA-757, and two experimental chicken lines, specific pathogen free (SPF) layers and broilers, were infected at 2 days of age. Investigations focused on faecal shedding, bacterial colonization, humoral and cellular immune response. Ross and SPF broilers proved mainly as high shedders followed by Hubbard. SPF layers showed the least shedding. This is in agreement with the caecal colonization; SPF layers harboured significantly less bacteria. Systemic spread of S. Infantis to liver and spleen was highest in Ross broilers compared to the other lines. Spread of infection to in-contact birds, was noticed 5 days post infection in every line. Antibody response occurred in every chicken line from 21 days of age onwards. In contrast to the other chicken lines, significant differences in T cell subsets and monocytes/macrophages were found between infected and negative Hubbard birds at 7 days of age. Uninfected SPF birds had significantly higher immune cell counts than uninfected commercial birds, a fact important for future experimental settings. The results illustrate that the infection dynamics of S. Infantis is influenced by the chicken line resulting in a higher risk of transmission to humans from fast-growing broilers. RESEARCH HIGHLIGHTS Infection dynamics of Salmonella Infantis differs between chicken lines. Layers showed less faecal shedding and caecal colonization compared to broilers. Fast-growing broilers proved more susceptible than slow-growing broilers.

Gender differences and pharmacological regulation of angiogenesis induced by synovial fluids in inflammatory arthritis.

Several mediators including cytokines, growth factors and metalloproteinases (MMP) modulate pathological angiogenesis associated with inflammatory arthritis. The biological factors underlying sex disparities in the incidence and severity of rheumatic musculoskeletal diseases are only partially understood. We hypothesized that synovial fluids (SFs) from rheumatoid arthritis (RA) and psoriatic arthritis (PsA) patients would impact on endothelial biology in a sexually dimorphic fashion. Immune cell counts and levels of pro-angiogenic cytokines found in SFs from RA and PsA patients (n=17) were higher than in osteoarthritis patients (n=6). Synovial VEGF concentration was significantly higher in male than in female RA patients. Zymography revealed that SFs comprised solely MMP-9 and MMP-2, with significantly higher MMP-9 levels in male than female RA patients. Using in vitro approaches that mimic the major steps of the angiogenic process, SFs from RA and PsA patients induced endothelial migration and formation of capillary-like structures compared to control. Notably, endothelial cells from female donors displayed enhanced angiogenic response to SFs with respect to males. Treatment with the established anti-angiogenic agent digitoxin prevented activation of focal adhesion kinase and SF-induced in vitro angiogenesis. Thus, despite higher synovial VEGF and MMP-9 levels in male patients, the responsiveness of vascular endothelium to SF priming was higher in females, suggesting that gender differences in angiogenic responses were mainly related to the endothelial genotype. These findings may have implications for pathogenesis and targeted therapies of inflammatory arthritis.

Immunotherapeutic potential of ethanolic olive leaves extract (EOLE) and IL-28B combination therapy in ENU induced animal model of leukemia.

Cytokine therapies have shown promising results against cancers. Cytokines are secreted naturally from different bodily cells. These have fewer side effects but higher specificity than chemotherapy and radiation therapy. In leukemia, changes in normal hematopoiesis and defective leukocyte production limit the efficacy of immunotherapy by reducing the count of functional immune cells. Therefore, the treatment of leukemia needs advanced therapeutics that can target multiple cancer sustaining mechanisms. In combination therapy, using two different therapeutic agents affect cancer growth in many ways and sometimes gives synergistic effects. Here, we examined the effect of the ethanolic olive leaf extract (EOLE) and IL-28B in combination. N-N' Ethyl-nitrosourea (ENU) induced leukemia in Swiss albino mice was treated with EOLE for four weeks and IL-28B for one week after confirming the development of leukemia. The combination of EOLE and IL-28B significantly reduced the blast cell and total WBC counts in the peripheral blood, altered the levels of various cytokines in plasma, and induced the functional activity of NK cells in leukemic mice. The induced NK activity correlates with increased expression of perforin and granzyme studied at the gene level through real-time (RT)-PCR. The treatment of leukemic mice with combined EOLE and IL-28B has also caused an increased serum IL-10 and IFN-γ level, and reduced serum TGF-β indicates improved overall immunity. Altogether, the combination of EOLE and IL-28B has given substantial therapeutic activity against leukemia.

FK506 increases susceptibility to musculoskeletal infection in a rodent model

BackgroundDelayed fracture healing caused by soft tissue loss can be resolved by the administration of a Th1 immunosuppressant, such as FK506. Additionally, open fractures are at high risk for infection. We hypothesized that the inclusion of an immunosuppressant to a subject at risk for a musculoskeletal infection will increase the likelihood of infection.MethodsA rat model of musculoskeletal infection was used. Sprague Dawley rats received a stabilized femur defect and were inoculated with 104 CFU Staphylococcus aureus via a collagen matrix. Six hours after inoculation, the wounds were debrided of collagen and devitalized tissue and irrigated with sterile saline. The animals were randomized into two groups: carrier control and FK506, which were administered daily for 14 days and were euthanized and the tissues harvested to measure local bioburden.ResultsThe dosing regimen of FK506 that restored bone healing increased the bioburden in the bone and on the fixation implant compared to the carrier control animals. As expected, the administration of FK506 decreased circulating white blood cells, lymphocytes, neutrophils, and monocytes. Additionally, the red blood cell count, hematocrit, and body weight were lower in those animals that received FK506 compared to carrier control.ConclusionsFK506 administration decreased the systemic immune cell counts and increased the bacterial bioburden within a model of musculoskeletal infection. Collectively, these outcomes could be attributed to the overall T cell suppression by FK506 and the altered antimicrobial activity of innate cells, thereby allowing S. aureus to thrive and subsequently leading to infection of severe, musculoskeletal injuries. These observations reveal the crucial continued investigation for the clinical use of FK506, and other immunosuppressant compounds, in trauma patients who are at increased risk of developing infections.

Secondary Immune Deficiency and Primary Immune Deficiency Crossovers: Hematological Malignancies and Autoimmune Diseases.

Primary immunodeficiencies (PIDs), a heterogenous group of inborn errors of immunity, are predetermined at birth but may evolve with age, leading to a variable clinical and laboratory presentation. In contrast, secondary immunodeficiencies (SIDs) are acquired declines of immune cell counts and or/function. The most common type of SID is a decreased antibody level occurring as a consequence of extrinsic influences, such as an underlying condition or a side effect of some medications used to treat hematological malignancies and autoimmune disorders. Paradoxically, immune deficiencies initially attributed to secondary causes may partly be due to an underlying PID. Therefore, in the era of immune-modulating biologicals, distinguishing between primary and secondary antibody deficiencies is of great importance. It can be difficult to unravel the relationship between PID, SID and hematological malignancy or autoimmunity in the clinical setting. This review explores SID and PID crossovers and discusses challenges to diagnosis and treatment strategies. The case of an immunodeficient patient with follicular lymphoma treated with rituximab illustrates how SID in the setting of hematological cancer can mask an underlying PID, and highlights the importance of screening such patients. The risk of hematological cancer is increased in PID: for example, lymphomas in PID may be driven by infections such as Epstein-Barr virus, and germline mutations associated with PID are enriched among patients with diffuse large B-cell lymphoma. Clues suggesting an increased risk of hematological malignancy in patients with common variable immune deficiency (CVID) are provided, as well as pointers for distinguishing PID versus SID in lymphoma patients. Two cases of patients with autoimmune disorders illustrate how an apparent rituximab-induced antibody deficiency can be connected to an underlying PID. We highlight that PID is increasingly recognized among patients with autoimmune cytopenias, and provide guidance on how to identify PID and distinguish it from SID in such patients. Overall, healthcare professionals encountering patients with malignancy and/or autoimmunity who have post-treatment complications of antibody deficiencies or other immune abnormalities need to be aware of the possibility of PID or SID and how to differentiate them.

The Blood Immune Cell Count, Immunoglobulin, Inflammatory Factor, and Milk Trace Element in Transition Cows and Calves Were Altered by Increasing the Dietary n-3 or n-6 Polyunsaturated Fatty Acid Levels.

Transition dairy cows experience sudden changes in both metabolic and immune functions, which lead to many diseases in postpartum cows. Therefore, it is crucial to monitor and guarantee the nutritional and healthy status of transition cows. The objective of this study was to determine the effect of diet enriched in n-3 or n-6 polyunsaturated fatty acid (PUFA) on colostrum composition and blood immune index of multiparous Holstein cows and neonatal calves during the transition period. Forty-five multiparous Holstein dairy cows at 240 days of pregnancy were randomly assigned to receive 1 of 3 isoenergetic and isoprotein diets: 1) CON, hydrogenated fatty acid (control), 1% of hydrogenated fatty acid [diet dry matter (DM) basis] during prepartum and postpartum, respectively; 2) HN3, 3.5% of extruding flaxseed (diet DM basis, n-3 PUFA source); 3) HN6, 8% of extruding soybeans (diet DM basis, C18:2n-6 PUFA source). Diets containing n-3 and n-6 PUFA sources decreased colostrum immunoglobulin G (IgG) concentration but did not significantly change the colostrum IgG yield compared with those with CON. The commercial milk yield (from 14 to 28 days after calving) was higher in the HN3 and HN6 than that in the CON. Furthermore, the n-3 PUFA source increased neutrophil cell counts in blood during the prepartum period and increased neutrophil percentage during the postpartum period when compared with those with control treatment. Diets containing supplemental n-3 PUFA decreased the serum concentration of interleukin (IL)-1β in maternal cows compared with those in control and n-6 PUFA during prepartum and postpartum. In addition, the neonatal calf serum concentration of tumor necrosis factor (TNF) was decreased in HN3 compared with that in the HN6 treatment. The diet with the n-3 PUFA source could potentially increase the capacity of neutrophils to defend against pathogens in maternal cows by increasing the neutrophil numbers and percentage during the transition period. Meanwhile, the diet with n-3 PUFA source could decrease the pro-inflammatary cytokine IL-1β of maternal cows during the transition period and decline the content of pro-inflammatary cytokine TNF of neonatal calves. It suggested that the highest milk production in n-3 PUFA treatment may partially be due to these beneficial alterations.

Abstract 5887: Association of ovarian tumor immune profiles with overall survival

Abstract Despite being immunogenic, high-grade serous ovarian carcinomas (HGSC), the most common histotype, generally are not responsive to immune checkpoint inhibitors. To understand potential reasons for this, we examined associations of tumor-infiltrating B cells and T cells, including sub-populations defined by expression of immune checkpoints and T cell exhaustion markers, with survival in three epidemiologic studies. Analyses included participants in the Nurses’ Health Study (NHS), NHSII, and New England Case-control study who were diagnosed with stage 1-3 HGSC for whom tumor tissue was obtained. B cells (CD19, CD20, CD138), T cells (CD3), and T cell subpopulations (CD3 co-localized with CD8, CD4, PD1, PDL1, LAG3, TIM3) were measured using multiplex immunofluorescence assays performed on tissue microarray slides containing 3 cores per case. For each participant, we summed counts of immune cells infiltrating the tumor and total tumor cells across cores. Counts of immune cell subpopulations were adjusted for the parent cell count using residuals from beta-binomial models. Hazard ratios (HR) and 95% confidence intervals (CI) for overall mortality according to presence/absence or quantiles of immune cells were calculated using Cox proportional hazards regression models adjusting for age at diagnosis, diagnosis year, stage, study, and total tumor cell count. Among 389 women with HGSC, presence vs. absence of CD19+ B cells was suggestively associated with lower mortality (HR 0.79, 95%CI:0.61-1.01); similar associations were observed for CD19-adjusted CD20+ B cells and CD138+ plasma cells (HRs 0.75-0.78, quintile 5 vs. 1).Higher total T cells was associated with improved survival (HR 0.67, 95%CI:0.45-0.99, quintile 5vs. 1). Similar reduced risks were observed with high levels of both CD19+ B cells and CD3+ T cells (HR 0.66) or high levels of one cell type (HRs 0.65-0.75) compared with low levels of both. For the two primary T cell subfractions, lower risk was observed with higher CD3-adjustedCD3+CD8+ cytotoxic T cells (HRs 0.68, 0.73, 0.69, 0.59 for quintiles 2-5 vs. 1), but notCD3+CD4+ helper T cells (HR 0.97, 95%CI: 0.72-1.31, tertile 3 vs. 1). T cells with immune checkpoint markers (PD1, PDL1) and/or markers of T-cell exhaustion (LAG3, TIM3) generally were not associated with mortality or tended to be associated with lower mortality risk, with a significant inverse association observed for CD3-adjusted CD3+PD1+TIM3- cells (HR 0.61,95%CI:0.41-0.92, quintile 5 vs. 1).Overall, higher levels of B cells and CD3+CD8+ T cells in tumor were associated with improved outcomes, consistent with other studies. Congruent with the apparent ineffectiveness of immune checkpoint inhibitors in ovarian cancer, our results do not support that T cells expressing immune checkpoint and/or T cell exhaustion markers are related to worse outcome among women with HGSC. Future analyses will consider multiple cell types simultaneously and the stroma compartment. Citation Format: Shelley S. Tworoger, Mary K. Townsend, Jose Conejo-Garcia, Brooke L. Fridley, Carlos Moran Segura, Daryoush Saeed-Vafa, Naoko Sasamoto, Kathryn L. Terry. Association of ovarian tumor immune profiles with overall survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5887.

Abstract 449: Machine learning models identify histological features that can predict KEAP1 mutations in lung adenocarcinoma

Abstract Background: In lung cancer, the KEAP1/NRF2 pathway modulates an anti-tumor response by regulating cellular metabolism and inflammatory processes. Approximately 19% of lung adenocarcinoma (LUAD) tumors have a mutation in KEAP1. Clinically, KEAP1-mutated LUAD has poor prognosis, and there is need for rapid and accurate patient genotyping to inform treatment decisions. Here, we describe machine learning (ML) models that can predict KEAP1 mutation status from tissue histology. Methods: ML models, pre-trained to identify and quantify areas of tissue (cancer epithelium, cancer stroma, and necrosis), counts of cancer, fibroblast, and immune cells (lymphocytes, macrophages, plasma cells) in non-small cell lung cancer (NSCLC), were applied to 208 hematoxylin and eosin (H&E)-stained whole slide images (WSI) of LUAD from The Cancer Genome Atlas (TCGA) without further training. Genomic analyses indicated that 17% (N=35) of these cases are KEAP1MUT. Human Interpretable Features (HIFs), based on histology predictions, are automatically extracted from the model and provide a quantitative description of the tumor microenvironment of each WSI. Associations between HIFs and KEAP1MUT were determined by univariate analysis followed by false discovery rate (FDR) correction. Hierarchical clustering using cross correlation and combining p-values for HIF groups using an Empirical Brown’s method identified correlations between HIFs. Confounding factor influence was accounted for after positive associations were identified. Independent validation of associations between KEAP1MUT and HIFs was performed using TCGA transcriptomic data to correlate specific mutations with mRNA expression of relevant markers. Results: ML-models generated 4,443 HIFs from the TCGA LUAD WSI, which were reduced to 2,684 HIFs after removal of outlier HIFs, exclusion of HIFs that are degenerate, have missing features, or are of an absolute value. KEAP1MUT was significantly associated with 193 HIFs in univariate analyses (p<0.05 after FDR correction), and four groups of HIFs after taking correlations into account (p<0.05 after group-wise FDR correction; 211-264 HIFs per group). 161 HIFs were identified by both methods. Further assessment of the KEAP1MUT -associated HIFs showed that these mutations were correlated with a reduction in macrophages in the tumor microenvironment (TME). This was supported by analysis of transcriptomic data from KEAP1MUT (N=35) and KEAP1WT (N=164) samples, which showed significantly reduced expression of the macrophage marker CD14 (p<0.001) in KEAP1MUT tissue samples. Conclusions: ML model quantification of TME histological features can generate HIFs that correlate with the KEAP1MUT status of a LUAD biopsy. These results exemplify how ML-powered digital pathology could predict molecular markers directly from standard H&E biopsy slides. Citation Format: Robert Egger, Martin Ieong, Murray Resnick, Amaro Taylor-Weiner, Victoria Mountain, Ilan Wapinski, Michael Montalto, Andrew Beck, Josie Hayes, Benjamin Glass. Machine learning models identify histological features that can predict KEAP1 mutations in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 449.

The Effects of Cannabinoid Agonist, Heat Shock Protein 90 and Nitric Oxide Synthase Inhibitors on Increasing IL-13 and IL-31 Levels in Chronic Pruritus

ABSTRACT Background Heat shock protein 90 (Hsp90) inhibitor and cannabinoid agonists ameliorate dry skin-induced chronic itch. We have recently reported that cannabinoids, hsp90 and nitric oxide (NO) are involved in dry skin-induced itch. Here, we investigated the contribution of the Th2 cell signaling pathway to the antipruritic effect of the hsp90 inhibitor 17-Alilamino-17-demethoxygeldanamycin (17-AAG), nitric oxide synthase (NOS) inhibitor Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) and cannabinoid agonist WIN 55,212–2 on a dry skin-induced scratch. Methods Dry skin-induced chronic itching was created by topical application of AEW (acetone/diethyl ether/water). WIN 55,212–2 (1 mg/kg, i.p.), L-NAME (1 mg/kg, i.p.) and increasing doses of 17-AAG (1, 3 and 5 mg/kg,i.p.) were administered to Balb/c mice (for each group, n = 6). After these applications, skin tissues were taken from the nape region of all of the mice. Gene and protein expressions of IL-13 and IL-31 were evaluated in skin tissues by RT-PCR and immunohistochemistry, respectively. Results IL-13 and IL-31 mRNA expressions and immune positive cell counts were increased in the AEW applied groups. WIN 55,212–2 reduced both of the increased cytokines levels, while L-NAME decreased only the IL-13. 17-AAG dose-dependently reduced the increased cytokine levels. IL-13 and IL-31 levels significantly decreased following the co-administration of these agents. Conclusion These results show that increased levels of IL-13 and IL-31 are associated with pruritus. Hsp90 inhibition and cannabinoid system activation may induce antipruritic effects through down-regulation of these cytokines.

Exploratory analysis on crosstalk between intra-tumor immunity and FGF/FGFR pathway in clear cell renal cell carcinoma.

e16525 Background: Clear cell renal cell carcinoma (ccRCC) patients classically show good response to VEGFR inhibitors; however, the biological basis by which FGFR inhibition provides tumor response was not well understood. Here, we performed an exploratory analysis of the relationship between the FGF/FGFR pathway and intra-tumor immunity to support the effectiveness of FGFR inhibitors for ccRCC. Methods: We used whole transcriptome data and immunohistochemistry with metastatic clear cell RCC specimens from patients who were treated at the Yamagata University Hospital (n = 57). FFPE samples were prepared from the primary lesions of treatment-naive metastatic ccRCC patients. FFPE was sliced by a pathologist to evaluate the immunological microenvironment. RNA extraction and immunostaining were performed at the same site. Immuno-histological quantification was performed by automatically analysis system HALO in digital whole slide imaging. Hierarchical clustering was performed, and the cohort was divided based on the profile of the FGF and immune cells. We analyzed the cancer-specific survival based on the FGF cluster classification and investigated the relationship between FGF-FGFR expression and immune cell infiltration that were evaluated histologically. Results: RNA transcriptome data showed that FGF2 and FGF7 were widely expressed in ccRCC. Hierarchical cluster analysis based on the FGF expression profile (intrinsic FGFs were excluded) revealed that metastatic ccRCC had three distinct patterns. Cluster1 showed low FGFs expression, except for FGF2 and 7, while Cluster2 and 3 expressed multiple FGFs. Survival analysis showed Cluster2and 3 had a poor cancer-specific survival rate than Cluster1 (Logrank test, p = 0.05). However, no association was detected between the FGF cluster classification and the infiltration of immune cell count in immunohistological analysis. In comparison of the expression of each FGF subfamily and the positive cells of CD4, CD8, CD68 and CD31 (Mann–Whitney U test, p < 0.05), CD4 and CD8 were higher in the cases expressing FGF20. While most FGF subfamilies have been suggested to be less associated with immune cell infiltration (excluding FGF20), further analysis among FGF receptor expression and immune signatures are ongoing. Conclusions: Our study demonstrates the possibility of intra-tumor interaction between FGF/FGFR axis and immune infiltration in ccRCC.

Blood monocyte and lymphocyte counts patient outcomes in non-small cell lung cancer after immune checkpoint inhibitor therapy: Real-world data of 12,416 patients.

e21023 Background: Immune checkpoint inhibitors (ICPi) are now commonly used in non-small cell lung cancer (NSCLC) treatment. Beyond PD-L1 status, no clinically validated predictive biomarkers guide therapeutic decision making regarding their use. Blood monocyte, lymphocyte counts, or their ratio might inform clinical utility of these expensive, potentially toxic agents. Methods: Adults with advanced/metastatic NSCLC receiving any ICPi after diagnosis from January 2015 to November 2020 were included. Treatment outcomes were followed until 31 May 2021. Data were sourced from structured fields of electronic health records from The US Oncology Network and non-Network practices. The primary end point was overall survival (OS), and time to treatment discontinuation (TTD) was secondary. Baseline absolute monocyte count (AMC), absolute lymphocyte count (ALC), and monocyte-to-lymphocyte ratio (MLR) were subdivided into quintiles (Q) with Q1 lowest and Q5 highest. Patient (pt) and treatment characteristics were described. Time-to-event outcomes were assessed by Kaplan-Meier methods. Univariate and multivariate Cox regression analyses described associations among baseline biomarkers, OS, and TTD. Results: For 12,416 pts with NSCLC, median OS and TTD were 13.6 months (mos) and 4.5 mos, respectively. For AMC, patients in Q2 and Q3 had the longest median OS of 15.9 mos. Median TTD by AMC was longest for Q1 and Q2 (5.1 mos), declining sequentially to 3.3 mos for Q5. For ALC, Q5 had longest median OS of 19.2 mos, as well as longest median TTD of 5.8 mos. For MLR, Q1 had the longest median OS and TTD of 22.3 and 6.2 mos., respectively. In univariate Cox regression, AMC, ALC, and MLR each associated with OS and TTD (p < 0.0001). Pts in AMC Q5, compared with AMC Q1, had an OS hazard ratio (HR) of 1.48 (95% CI 1.38-1.60). For ALC, each successive increasing quintile, compared to ALC Q1, demonstrated a lower risk of death (p < 0.0001). Pts in MLR Q5, compared with MLR Q1, had an OS HR of 2.21 (95% CI 2.04-2.39). Conclusions: This data set is the largest to date to examine interactions among baseline immune cell counts, ICPi use, and pt outcome in NSCLC. These data inform efforts to describe immune-based prognostic risk groups among NSCLC pts receiving immunotherapy.[Table: see text]

Advantages and Challenges of Differential Immune Cell Count Determination in Blood and Milk for Monitoring the Health and Well-Being of Dairy Cows

A key challenge of the 21st century will be to provide the growing world population with a sustainable and secure supply of food. Consequently, the dairy farming’s primary task is to lower milk losses and other inefficiencies associated with diseased cows. Moreover, a shift from curative to preventive health management would be desirable for mastitis and a wide variety of other infectious and non-infectious cattle diseases, some of which are known to have profound negative effects on the performance and well-being of cows. Differential cell counting (DCC), a procedure that aims to determine the proportions of different somatic cell types in raw milk samples, has not only the potential to optimize mastitis diagnostics, but it could furthermore serve as a diagnostic tool for monitoring the general and overall health status of dairy cows. Based on a broad search of the literature, the practical utility of various types of DCC is summarized and discussed in this review. Since it might be of advantage to interpret DCC with the aid of data from studies in humans, differences between the immune systems of humans and dairy cattle, with a special focus on surface marker expression profiles and γδ (gamma delta) T-cell characteristics, are also described.