Granulocyte Research Articles

Disruption of the Functional Activity of Neutrophil Granulocytes as a Risk Factor for the Development of Lung Damage in Pregnant Women with COVID-19.

Currently, the assessment of immune status in patients with COVID-19 is limited to determining the count of polymorphonuclear leukocytes and the phagocytic function of neutrophils, which is insufficient to understand the regulatory role of innate immunity cells in the development of pneumonia. However, no such studies have been conducted in pregnant women with COVID-19. The aim of this study was to investigate the functional state of neutrophil granulocytes in order to identify predictors of pneumonia severity risk in pregnant women with COVID-19. A clinical characterization of pregnant women with COVID-19 in addition to minimal and average lung changes was provided. The composition and ratio of morphological forms of leukocyte cells were studied. Cytochemical studies of neutrophil granulocytes were carried out and calculations of the mean cytological index (MCI) for succinate dehydrogenase, myeloperoxidase, and cationic proteins were performed. The number of NETs in blood smears was counted. Independent predictors of pneumonia severity in pregnant women with COVID-19 were calculated using regression analysis. The quality of the model was assessed using ROC analysis. In pregnant women with COVID-19 and an average volume of lung changes, the number of polymorphonuclear leukocytes (p = 0.03) and band neutrophils (p = 0.002) in the blood was significantly higher than in pregnant women with minimal lung changes. The MCI indicators of succinate dehydrogenase, cationic proteins, and myeloperoxidase in pregnant women with COVID-19 were reduced in relation to the control group (p < 0.0001). In blood smears of pregnant women with COVID-19 and an average volume of lung changes, the number of NETs increased (p = 0.002). Regression analysis showed that succinate dehydrogenase and NETs are independent predictors of pneumonia severity in pregnant women with COVID-19. Our study confirms the prognostic significance of low levels of neutrophilic succinate dehydrogenase and high levels of NETs in the blood of pregnant women with COVID-19. The combination of these two biomarkers is a significant reflection of the severity of pneumonia development in pregnant women with COVID-19. However, further research is needed to identify the mechanisms underlying this association.

Hypomethylation at H19DMR in penile squamous cell carcinoma is not related to HPV infection

ABSTRACT Penile squamous cell carcinoma (SCC) is a rare and aggressive tumour mainly related to lifestyle behaviour and human papillomavirus (HPV) infection. Environmentally induced loss of imprinting (LOI) at the H19 differentially methylated region (H19DMR) is associated with many cancers in the early events of tumorigenesis and may be involved in the pathogenesis of penile SCC. We sought to evaluate the DNA methylation pattern at H19DMR and its association with HPV infection in men with penile SCC by bisulfite sequencing (bis-seq). We observed an average methylation of 32.2% ± 11.6% at the H19DMR of penile SCC and did not observe an association between the p16INK4a+ (p = 0.59) and high-risk HPV+ (p = 0.338) markers with methylation level. The average methylation did not change according to HPV positive for p16INK4a+ or hrHPV+ (35.4% ± 10%) and negative for both markers (32.4% ± 10.1%) groups. As the region analysed has a binding site for the CTCF protein, the hypomethylation at the surrounding CpG sites might alter its insulator function. In addition, there was a positive correlation between intense polymorphonuclear cell infiltration and hypomethylation at H19DMR (p = 0.035). Here, we report that hypomethylation at H19DMR in penile SCC might contribute to tumour progression and aggressiveness regardless of HPV infection.

Genetic parameters for novel mastitis traits defined by combining test-day somatic cell score and differential somatic cell count in the first lactation of Japanese Holsteins

In this study, we aimed to improve current udder health genetic evaluations by addressing the limitations of monthly sampled somatic cell score (SCS) for distinguishing cows with robust innate immunity from those susceptible to chronic infections. The objectives were to (1) establish novel somatic cell traits by integrating SCS and the differential somatic cell count (DSCC), which represents the combined proportion of polymorphonuclear leukocytes and lymphocytes in somatic cells and (2) estimate genetic parameters for the new traits, including their daily heritability and genetic correlations with milk production traits and SCS, using a random regression test-day model (RRTDM). We derived 3 traits, namely ML_SCS_DSCC, SCS_4_DSCC_65_binary, and ML_SCS_DSCC_binary, by using milk loss estimates at corresponding SCS and DSCC levels, thresholds established in previous studies, and a threshold established from milk loss estimates, respectively. Data consisted of test-day records collected during January 2021 through March 2022 from 265 herds in Hokkaido, Japan. From these records, we extracted records between 7 to 305 d in milk (DIM) in the first lactation to fit the RRTDM. The model included the random effect of herd-test-day, the fixed effect of year-month, fixed lactation curves nested with calving age groups, and random regressions with Legendre polynomials of order 3 for additive genetic and permanent environmental effects. The analysis was performed using Gibbs sampling with Gibbsf90+ software. The averages (ranges) of daily heritability estimates over lactation were 0.086 (0.075 to 0.095) for SCS, 0.104 (0.073 to 0.127) for ML_SCS_DSCC, 0.137 (0.014 to 0.297) for SCS_4_DSCC_65_binary, and 0.138 (0.115 to 0.185) for ML_SCS_DSCC_binary; the heritability curve for SCS_4_DSCC_65_binary was erratic. Genetic correlations within the trait decreased as the DIM interval widened, especially for those integrating DSCC, indicating that these traits should be analyzed using RRTDM rather than repeatability models. The averages (ranges) of genetic correlations with milk yield over lactation were 0.01 (-0.22 to 0.28) for SCS, -0.05 (-0.40 to 0.13) for ML_SCS_DSCC, -0.08 (-0.17 to 0.09) for SCS_4_DSCC_65_binary, and -0.08 (-0.22 to 0.27) for ML_SCS_DSCC_binary. Compared with SCS, the newly defined traits exhibited slightly stronger negative genetic correlations with milk yield. Especially in late lactation stages, the genetic correlation between ML_SCS_DSCC and milk yield was significantly below zero, with a posterior median of -0.40. Furthermore, the new traits showed positive correlations with SCS, having estimates varying from 0.68 to 0.85 for ML_SCS_DSCC, 0.14 to 0.47 for SCS_4_DSCC_65_binary, and 0.61 to 0.66 for ML_SCS_DSCC_binary, depending on DIM. Considering that ML_SCS_DSCC and ML_SCS_DSCC_binary have relatively high heritability (compared with SCS) and favorable genetic correlations with milk production traits and SCS, their incorporation into breeding programs appears promising. Nevertheless, their genetic relationships with (sub)clinical mastitis require further investigation.

Surface CD52, CD84, and PTGER2 mark mature PMN-MDSCs from cancer patients and G-CSF-treated donors

Precise molecular characterization of circulating polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) is hampered by their mixed composition of mature and immature cells and lack of specific markers. Here, we focus on mature CD66b+CD10+CD16+CD11b+ PMN-MDSCs (mPMN-MDSCs) from either cancer patients or healthy donors receiving G-CSF for stem cell mobilization (GDs). By RNA sequencing (RNA-seq) experiments, we report the identification of a distinct gene signature shared by the different mPMN-MDSC populations under investigation, also validated in mPMN-MDSCs from GDs and tumor-associated neutrophils (TANs) by single-cell RNA-seq (scRNA-seq) experiments. Analysis of such a gene signature uncovers a specific transcriptional program associated with mPMN-MDSC differentiation and allows us to identify that, in patients with either solid or hematologic tumors and in GDs, CD52, CD84, and prostaglandin E receptor 2 (PTGER2) represent potential mPMN-MDSC-associated markers. Altogether, our findings indicate that mature PMN-MDSCs distinctively undergo specific reprogramming during differentiation and lay the groundwork for selective immunomonitoring, and eventually targeting, of mature PMN-MDSCs.

Machine Learning-assisted immunophenotyping of peripheral blood identifies innate immune cells as best predictor of response to induction chemo-immunotherapy in head and neck squamous cell carcinoma – knowledge obtained from the CheckRad-CD8 trial

PurposeIndividual prediction of treatment response is crucial for personalized treatment in multimodal approaches against head-and-neck squamous cell carcinoma (HNSCC). So far, no reliable predictive parameters for treatment schemes containing immunotherapy have been identified. This study aims to predict treatment response to induction chemo-immunotherapy based on the peripheral blood immune status in patients with locally advanced HNSCC. MethodsThe peripheral blood immune phenotype was assessed in whole blood samples in patients treated in the phase II CheckRad-CD8 trial as part of the pre-planned translational research program. Blood samples were analyzed by multicolor flow cytometry before (T1) and after (T2) induction chemo-immunotherapy with cisplatin/docetaxel/durvalumab/tremelimumab. Machine Learning techniques were used to predict pathological complete response (pCR) after induction therapy. ResultsThe tested classifier methods (LDA, SVM, LR, RF, DT, and XGBoost) allowed a distinct prediction of pCR. Highest accuracy was achieved with a low number of features represented as principal components. Immune parameters obtained from the absolute difference (lT2-T1l) allowed the best prediction of pCR. In general, less than 30 parameters and at most 10 principal components were needed for highly accurate predictions. Across several datasets, cells of the innate immune system such as polymorphonuclear cells, monocytes, and plasmacytoid dendritic cells are most prominent. ConclusionsOur analyses imply that alterations of the innate immune cell distribution in the peripheral blood following induction chemo-immuno-therapy is highly predictive for pCR in HNSCC.

Low-laser action analysis associated with Himatanthus drasticus extract in wound healing.

The aim of this study was to analyze the healing action of low-intensity laser therapy associated with Himatanthus drasticus in wound healing in mice. The study was experimental, analytical, controlled, randomized, and prospective. Twenty Wistar rats were divided into four groups: control with injury, injury + laser, wound + Himatanthus drasticus, and wound + laser + Himatanthus drasticus. The animals underwent surgical skin wounds on their backs, and different treatments were applied based on the group. The healing process was evaluated histologically through the analysis of collagen fibers, neovascularization, and inflammatory reaction. The results showed that low-intensity laser therapy and Himatanthus drasticus treatment improved the healing process in terms of collagen synthesis, decreased inflammatory cell migration, fibroblast proliferation, neovascularization, wound size reduction, and edema reduction. However, the combination of laser and Himatanthus drasticus did not show significant improvement compared to individual treatments. There were no statistical differences in polymorphonuclear cells between the treatment groups. Low-intensity laser therapy and Himatanthus drasticus have demonstrated positive properties in improving the healing process. Further research is needed to better understand their individual and combined effects. The study contributes to the exploration of alternative wound healing methods and encourages further investigation in this field.

Abstract B019: Tumor-derived PTHrP molds an immunosuppressive microenvironment

Abstract Purpose[JP1] : Molecular phenotyping of PDAC [PJ2] has revealed an aggressive squamous/basal/quasi-mesenchymal[JP3] subtype, clinically presenting as highly metastatic and harboring genetic amplifications of KRAS. Our previous work indicated that a squamous identity gene, Parathyroid Hormone Like Hormone (PTHLH; encoding for the PTHrP protein), is frequently co-amplified with KRAS and skews PDAC towards a squamous identity. Further, PTHrP is secreted by tumor cells and promotes tumor growth and metastasis through autocrine signaling. However, pathological analysis of PTHrP-null tumors indicated that the tumor microenvironment (TME) was re-organized upon loss of PTHrP. Thus, we hypothesized that PTHrP is a tumor-derived factor that shapes the TME through its systemic secretion and mobilization of immunosuppressive myeloid cells. Methods: In vivo experiments used KrasG12D; p53R172H; Pdx1-Cre; YFP (KPCY) and KPCY-PthlhloxP/WT (KPCY-Pthlhhet) for spontaneous tumor formation in immune competent mice. Orthotopic implantation experiments used Pthlh overexpressing KPCY tumor cells (herein KPCY-PthlhOE) or empty vector control cells (KPCY-PthlhEV). Tumor immunophenotyping was done using flow cytometry. Cytokine expression was performed using qPCR and ELISA. Results: In vivo investigation of tumors from KPCY-Pthlhhet mice revealed less MDSC and greater CD8+ T cell infiltration than KPCY controls, demonstrating the necessity of PTHrP for immunosuppression[PJ4]. Orthotopic implantation of KPCY-PthlhOE cell lines reduced overall TME immune cell recruitment, while simultaneously increasing the proportion of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) infiltration. A concomitant decrease in the frequency of T cells in the KPCY-PthlhOE tumors was also observed, showing the sufficiency of PTHrP to organize an immunosuppressive TME[PJ5]. Additionally, a reduction of PD-1+ and CD44high CD8+ T cells revealed they were less activated. Interestingly, recombinant human PTHrP was sufficient to induce MDSC mobilization in vivo.[JP6] To interrogate the mechanism of MDSC trafficking into the TME, cytokine production by was analyzed in vitro. KPCY-PthlhOE exhibited a pronounced increase and decrease of Cxcl1 and Cxcl10 expression, respectively, relative to the KPCY-EV control. Conclusions: The work herein describes a new mechanism for the mobilization and infiltration of immune suppressive MDSC in the tumor microenvironment, initiated by PTHrP. The PTHrP-dependent immunosuppressive TME excludes and inhibits effector immune cells, such as CD8+ T cells. This phenomenon has been described in squamous-like PDAC but may also apply to other squamous adenocarcinomas. [PJ7] Further elucidation of the mechanism by which PTHrP is the genesis of the immune suppressive TME may spur the development of novel treatments for these more clinically severe cancer subtypes. Citation Format: Calvin P. Johnson, Boyang Ma, Jason Pitarresi. Tumor-derived PTHrP molds an immunosuppressive microenvironment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B019.

Alpha-7 Nicotinic Receptor Agonist Protects Mice Against Pulmonary Emphysema Induced by Elastase.

Pulmonary emphysema is a primary component of chronic obstructive pulmonary disease (COPD), a life-threatening disorder characterized by lung inflammation and restricted airflow, primarily resulting from the destruction of small airways and alveolar walls. Cumulative evidence suggests that nicotinic receptors, especially the α7 subtype (α7nAChR), is required for anti-inflammatory cholinergic responses. We postulated that the stimulation of α7nAChR could offer therapeutic benefits in the context of pulmonary emphysema. To investigate this, we assessed the potential protective effects of PNU-282987, a selective α7nAChR agonist, using an experimental emphysema model. Male mice (C57BL/6) were submitted to a nasal instillation of porcine pancreatic elastase (PPE) (50µl, 0.667IU) to induce emphysema. Treatment with PNU-282987 (2.0mg/kg, ip) was performed pre and post-emphysema induction by measuring anti-inflammatory effects (inflammatory cells, cytokines) as well as anti-remodeling and anti-oxidant effects. Elastase-induced emphysema led to an increase in the number of α7nAChR-positive cells in the lungs. Notably, both groups treated with PNU-282987 (prior to and following emphysema induction) exhibited a significant decrease in the number of α7nAChR-positive cells. Furthermore, both groups treated with PNU-282987 demonstrated decreased levels of macrophages, IL-6, IL-1β, collagen, and elastic fiber deposition. Additionally, both groups exhibited reduced STAT3 phosphorylation and lower levels of SOCS3. Of particular note, in the post-treated group, PNU-282987 successfully attenuated alveolar enlargement, decreased IL-17 and TNF-α levels, and reduced the recruitment of polymorphonuclear cells to the lung parenchyma. Significantly, it is worth noting that MLA, an antagonist of α7nAChR, counteracted the protective effects of PNU-282987 in relation to certain crucial inflammatory parameters. In summary, these findings unequivocally demonstrate the protective abilities of α7nAChR against elastase-induced emphysema, strongly supporting α7nAChR as a pivotal therapeutic target for ameliorating pulmonary emphysema.

Age-related exacerbation of lung damage after trauma is associated with increased expression of inflammasome components.

Trauma, a significant global cause of mortality and disability, often leads to fractures and hemorrhagic shock, initiating an exaggerated inflammatory response, which harms distant organs, particularly the lungs. Elderly individuals are more vulnerable to immune dysregulation post-trauma, leading to heightened organ damage, infections, and poor health outcomes. This study investigates the role of NF-κB and inflammasomes in lung damage among aged mice post-trauma. Twelve male C57BL/6J mice underwent hemorrhagic shock and a femoral fracture (osteotomy) with external fixation (Fx) (trauma/hemorrhage, THFx), while another 12 underwent sham procedures. Mice from young (17-26 weeks) and aged (64-72 weeks) groups (n=6) were included. After 24h, lung injury was assessed by hematoxylin-eosin staining, prosurfactant protein C (SPC) levels, HMGB1, and Muc5ac qRT-PCR. Gene expression of Nlrp3 and Il-1β, and protein levels of IL-6 and IL-1β in lung tissue and bronchoalveolar lavage fluid were determined. Levels of lung-infiltrating polymorphonuclear leukocytes (PMNL) and activated caspase-3 expression to assess apoptosis, as well as NLRP3, ASC, and Gasdermin D (GSDMD) to assess the expression of inflammasome components were analyzed via immunostaining. To investigate the role of NF-κB signaling, protein expression of phosphorylated and non-phosphorylated p50 were determined by western blot. Muc5ac, and SPC as lung protective proteins, significantly declined in THFx versus sham. THFx-aged exhibited significantly lower SPC and higher HMGB1 levels versus THFx-young. THFx significantly increased activated caspase-3 versus both sham groups, and THFx-aged had significantly more caspase-3 positive cells versus THFx-young. IL-6 significantly increased in both sham and THFx-aged groups versus corresponding young groups. THFx significantly enhanced PMNL in both groups versus corresponding sham groups. This increase was further heightened in THFx-aged versus THFx-young. Expression of p50 and phosphorylated p50 increased in all aged groups, and THFx-induced p50 phosphorylation significantly increased in THFx-aged versus THFx-young. THFx increased the expression of inflammasome markers IL-1β, NLRP3, ASC and GSDMD versus sham, and aging further amplified these changes significantly. This study's findings suggest that the aging process exacerbates the excessive inflammatory response and damage to the lung following trauma. The underlying mechanisms are associated with enhanced activation of NF-κB and increased expression of inflammasome components.

Synovial tissue features associated with poor prognosis in inflammatory arthritis

BackgroundInflammatory arthritis encompasses a group of immune-mediated diseases characterized by chronic joint inflammation. Despite having pathogenic mechanisms in common, the prognosis of rheumatoid arthritis (RA), psoriatic arthritis (PsA), and undifferentiated arthritis (UA) could be different regarding progression to chronic, to erosive, or to self-limited disease. Our aim was to evaluate the potential association of synovial tissue (ST) inflammatory cell infiltrate, the presence of ectopic lymphoid neogenesis (LN +) structures, and poor prognosis factors (PPF) in patients with RA, PsA, and UA.MethodsWe conducted a retrospective study including patients with active arthritis (RA, PsA, UA) who had ST obtained by rheumatological arthroscopy or ultrasound-guided biopsy. Clinical, demographic, and immunohistochemical data of the synovium was evaluated. Patients with biological therapy at the time of synovial biopsy were excluded. PPF in patients with RA and UA were defined by the presence of anti-cyclic citrullinated peptide antibodies and/or rheumatoid factor, development of bone erosions, or requirement of biological therapy during the follow-up. PPF in patients with PsA were defined as the presence of high levels of acute-phase reactants (ESR/CRP), dactylitis or nail involvement at the time of biopsy, development of bone erosion, or requirement of biological therapy during the follow-up.ResultsA total of 88 patients were included: 26 RA, 33 PsA, and 29 UA. All patients were followed up for 5 years after the biopsy. Fourteen (53.84%) RA patients had PPF, and 17 (65.38%) had LN + . LN + was associated with PPF (p 0.038) and biologic therapy initiation (p 0.018). A total of 14 (43.75%) PsA patients had PPF. CD15 infiltrate (410.68 [SD 477.63] cells/mm2) was associated with PPF (p 0.008) in PsA patients. Sixteen (55.17%) patients with UA had PPF, and 13 (44.82%) had LN + . In this group, synovial CD68 + macrophages cells density was negatively correlated with DAS28-CRP (r = − 0.346, p 0.042).ConclusionsThe presence of LN + and higher CD15 + polymorphonuclear cells infiltrate was associated with PPF in RA and PsA, respectively. No associations were found for UA. These findings suggest a great heterogeneity of the ST features and its pathogenic implications in the subtypes of inflammatory arthritis.

Diagnostic Test Performances for Identifying Periprosthetic Joint Infection in Hip Hemiarthroplasty.

Periprosthetic joint infection (PJI) is a devastating complication of hip hemiarthroplasty (HHA) that is not well-represented in the literature. Therefore, this study aimed to evaluate diagnostic markers for identifying PJI in patients after HHA and compare them with the most recent 2018 International Consensus Meeting on Musculoskeletal Infection criteria. A total of 98 patients (64 PJIs, 65.3%) were analyzed. Patients were identified by relevant Current Procedural Terminology and International Classification of Diseases-9/10 codes from 2000 to 2021 across a single healthcare system. Preoperative or intraoperative synovial fluid nucleated cell (NC) count, synovial polymorphonuclear (PMN) percentage, serum erythrocyte sedimentation rate (ESR), serum C-reactive protein (CRP), and serum white blood cell count were compared with Student t -test between aseptic and septic cohorts. Diagnostic utility and laboratory cutoff values were determined using receiver-operating characteristic curves and Youden index, respectively. Mean values were significantly higher in the septic cohort for synovial NC count (120,992.2 versus 1,498.0 cells/μL, P < 0.001), synovial PMN percentage (91.3% versus 56.2%, P < 0.001), serum ESR (75.6 versus 36.3 mm/hr, P < 0.001), serum CRP (20.2 versus 125.8 mg/L, P < 0.001), and serum white blood cell count (8.5 versus 11.5 cells/μL, P < 0.001). Synovial NC count, synovial PMN percentage, and serum CRP had excellent PJI discriminatory ability with an area under the curve of 0.99, 0.90, and 0.93, respectively. Optimal cutoffs were 2,700 cells/μL for synovial NC count (100% sensitivity and 94% specificity), 81.0% for synovial PMN percentage (96% sensitivity and 89% specificity), 52.0 mm/hr for serum ESR (75% sensitivity and 80% specificity), and 40.0 mg/L for serum CRP (85% sensitivity and 92% specificity). Our findings support the continued use of routine serum and synovial fluid tests for diagnosing PJI in HHA patients. Optimal cutoff values for both synovial fluid biomarkers were very close in alignment with the 2018 International Consensus Meeting criteria. Level III, retrospective comparative study.

Aspergillus fumigatus-Derived Extracellular Vesicles Mitigate Fungal Keratitis by Modulating the Immune Cell Function.

Fungal keratitis (FK) is a refractory global disease characterized by a high incidence of blindness and a lack of effective therapeutic options, and Aspergillus fumigatus (A. fumigatus, AF) is one of the most common causative fungi. This study aimed to investigate the role of extracellular vesicles (EVs) from A. fumigatus in the immune cell function and their protective role in A. fumigatus keratitis in order to explore their therapeutic potential. First, we isolated and characterized the EVs (AF-derived EVs). In vitro, we stimulated RAW264.7 cells and polymorphonuclear cells with AF-derived EVs. The expression levels of inflammatory factors increased in both immune cells along with an M1 polarization variation of RAW264.7 cells. After being incubated with AF-derived EVs, both immune cells exhibited an increased conidia-phagocytic index and a decreased conidia survival rate. In vivo, we injected EVs subconjunctivally on mice resulting in a heightened production of secretory immunoglobulin A (sIgA) in tear fluid. By the injection of EVs on mice in advance, a significant reduction in severity of A. fumigatus FK was witnessed by lower clinical scores, inflammatory appearances, and mitigated fungal load. Collectively, these results positioned AF-derived EVs as a promising and innovative immune therapy for combating FK, while also providing a platform for further investigation into developing an optimal formulation for modulating inflammation in the context of FK.

Use of a commercial feed supplement based on diatom earth and yeast products on oxidative status and in vitro immune response in buffaloes during peripartum.

The transition period is a critical metabolic phase for dairy ruminants, especially those with high production levels. In spite of this, little is still known about dairy water buffalo. The aim of this study was to evaluate the effect of a commercial feed additive based on diatomaceous earth and hydrolyzed yeasts on health status, milk quality, and immune response of buffalo cows during the transition period. Eighty healthy Water buffaloes (Bubalus bubalis) of Italian Mediterranean breed were included in the trial. They were subdivided into two groups: one group received the additive (n = 40) while the control group (n = 40) received a placebo. The trial lasted 120d, from 60d before calving to 60d in milk. Blood samples were collected from each buffalo at -60 (60d from the expected calving), -30, 0 (calving), +15, +30, and +60 d (respectively, i.e., 15, 30, and 60d in milking). The biochemical as well as the oxidative profile, and the antioxidant power and enzymatic activity were evaluated in the samples obtained. Moreover, acute phase proteins, reactive proteins, and interleukin plasma levels were determined. Peripheral blood mononuclear cells (PBMCs) and monocytes were isolated and viability, reactive oxygen species (ROS), and reactive nitrogen species were measured on PBMC and monocytes. The introduction of additives enhanced the total antioxidant capacity and enzyme activity, while no differences were observed in oxidation products throughout the trial. Additionally, it significantly reduced the synthesis of ROS in polymorphonuclear cells, supporting a potential positive response in animals experiencing inflammation. The impact of oxidation on the products was not evident. Despite higher enzyme levels in plasma, this did not necessarily correspond to significantly increased enzymatic activity but rather indicated a higher potential. From these results, it was evident that the transition period in buffaloes differs notably from what reported in the literature for cows, probably due to the absence of common postpartum production diseases in dairy cows and lower metabolic challenges linked to lower milk production in buffaloes. Few parameters exhibited notable changes during the transition period in buffaloes, notably certain antioxidant enzymes, PBMC viability, PBMC ROS production, and Hp levels.

Combination of radiotherapy and Anlotinib enhances benefit from immunotherapy to liver metastasis and abscopal tumor from lung cancer

Many studies have shown that liver metastasis can weaken the efficacy of immunotherapy. Immunotherapy combined with radiotherapy or anti-angiogenic therapy has been proven to have synergistic anti-tumor effects. So we devote to explore whether the combination of the three therapies can exert effective anti-tumor effects on liver metastasis. The clinical information of 118 patients with liver metastasis were collected to compare the intrahepatic progression-free survival between immunotherapy and immunotherapy combined with other treatments. We used Lewis lung cancer (LLC) cell to establish a mouse liver metastasis tumor model and record tumor burden and survival. Tumor-infiltrating immune cells detected by flow cytometry. RNA sequencing was performed and the proportion of immune cells were analyzed by TIMER2.0 database. Compared with immunotherapy group, the combination therapy group showed a trend for longer median intrahepatic progression-free survival. Radiotherapy combined with PD-1 inhibitor and Anlotinib can inhibit liver metastasis and subcutaneous tumor growth and prolong the survival compared with other groups in vivo. Compared with the anti-PD-1 treatment group, triple therapy can increase CD4+T, CD8+T, and IFN-γ+CD8+T cells and decrease infiltration of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) in tumors. PPAR signaling pathway were significantly activated and CD8+T and dendritic cells (DC) were increased in the triple therapy group compared to the PD-1 inhibitor combined with Anlotinib group. Radiotherapy combined with PD-1 inhibitor and Anlotinib can effectively exert anti-tumor efficacy and reshape the tumor immune microenvironment by increasing the infiltration of anti-tumor immune cells and reducing the infiltration of immunosuppressive immune cells.

TREATMENT FOR TRANSMISSIBLE VENEREAL TUMOR IN FEMALE DOG WITH TUMOR EXCISION THROUGH EPISIOTOMY APPROACH

Transmission Venereal Tumor (TVT) is a malignant tumor that is contagious in dogs. Tumors are transmitted to other dogs generally through direct contact with dogs of sufferers during the mating season. A mixture of a female Rottweiler-Corgi mix breed called a 5-year-old Bocil, black and brown hair, with a body weight of 8.9 kg has a complaint of a lump in the vulva and genital swelling accompanied by the release of blood from the genitals. Routine hematology examination shows dogs experiencing thrombocytopenia. Histopathological examination of tumor tissue, indicates the presence of tumor cells in the form of lymphoblasts that experience modosis, and many hyperchromatic polymorphonuclear cells are found. Based on the history, clinical examination, and supporting examination, dogs are diagnosed with transmission venereal tumors. Surgery is carried out with the excision of the mass of the tumor through the episiotomy approach. Postoperatively given CefotaXime antibiotics at a dose of 20 mg/kg BW in IV Q12 hours for three days and analgesic dipyrone 25 mg/kg BW in IV q12 hours for 3 days, followed by giving Cefixime antibiotics 10 mg/kg BW twice four days and analgesic metamizole 25 mg/kg PO twice a day for 2 days. Dogs are given vincristine sulphate chemotherapy at a dose of 0.025 mg/kg BW intravenously every 1 time a week for three weeks in a row. On the 21st day postoperative sex size shrink and no blood is found from the vagina. Prevention of recurrent TVT in dogs needs to be minimized by performing ovariohysterectomy surgery.

Veterinarians exposed to inhaled anesthetic present chromosome damage, apoptosis and cell cycle changes.

This cross-sectional study evaluated, for the first time, DNA damage, viability, and cell death of lymphocytes and cell cycle phases of mononuclear and polymorphonuclear cells in veterinarians exposed to the volatile anesthetic isoflurane. Veterinarians who were occupationally exposed to isoflurane (exposed group; n = 20) and matched-unexposed individuals (volunteers without occupational exposure; n = 20) were enrolled in the study. DNA damage was assessed in lymphocytes by micronucleus (MN) and phosphorylated histone gamma-H2AX (γ-H2AX). Cell viability, cytotoxicity, and the cell cycle were evaluated by flow cytometry. Isoflurane was detected in urine samples by headspace gas chromatography-mass spectrometry. Compared with unexposed subjects, veterinarians occupationally exposed to isoflurane (25.7 ± 23.7 μg/L urine) presented statistically higher MN frequencies, lymphocytic apoptosis rates, and numbers of polymorphonuclear cells in the G0/G1 stage. Additionally, the exposed group presented statistically lower proportions of viable lymphocytes and G2/M polymorphonuclear cells. Our findings indicate that veterinarians who are frequently exposed to inhaled anesthetic exhibit chromosomal and cell damage in addition to changes in peripheral blood cell proliferation.

Helicobacter pylori infection and oxidative stress.

Helicobacter pylori (H. pylori) infection promotes the migration of polymorphonuclear leukocytes from the gastric mucosal microcirculation through chemokine induction, leading to the excessive production of ROS. Like eukaryotes, H. pylori possesses superoxide dismutase and catalase, and is resistant to ROS from host polymorphonuclear leukocytes. Oxidants such as monochloramine produced by ROS cause chronic inflammation in the gastric mucosa. H. pylori-derived virulence factor m1-type VacA induces intracellular ROS accumulation and autophagy, which degrades the H. pylori-derived oncoprotein, CagA. In CD44v9-positive gastric cancer stem-like cells, reduced-type glutathione levels increase within the cell because of the cystine transporter on the cell surface, wherein oxidative stress-induced autophagy no longer occurs. As a result, the oncoprotein CagA accumulates in the cells, thus becoming tumorigenic.

Flavonoids in mitigating the adverse effects of canine endotoxemia.

In dogs, chronic enteropathies, and impaired gut integrity, as well as microbiome imbalances, are a major problem. These conditions may represent a continuous low endotoxin load, which may result in the development of diseases that are attributable to chronic inflammation. Flavonoids are polyphenolic plant compounds with numerous beneficial properties such as antioxidant, anti-inflammatory and antimicrobial effects. For our experiments, we isolated primary white blood cells (peripheral blood mononuclear cells and polymorphonuclear leukocytes) from healthy dogs and induced inflammation and oxidative stress with Escherichia coli and Salmonella enterica serovar Enteritidis lipopolysaccharide (LPS). In parallel, we treated the cell cultures with various flavonoids luteolin, quercetin and grape seed extract oligomeric proanthocyanidins (GSOP) alone and also in combination with LPS treatments. Then, changes in viability, reactive oxygen species (ROS) and tumor necrosis factor alpha (TNF-α) levels were measured in response to treatment with quercetin, luteolin and GSOP at 25 and 50 μg/mL concentrations. We found that ROS levels were significantly lower in groups which were treated by flavonoid and LPS at the same time compared to LPS-treated groups, whereas TNF-α levels were significantly reduced only by luteolin and quercetin treatment. In contrast, treatment with lower concentrations of GSOP caused an increase in TNF-α levels, while higher concentrations caused a significant decrease. These results suggest that the use of quercetin, luteolin and GSOP may be helpful in the management of chronic intestinal diseases in dogs with reduced intestinal barrier integrity or altered microbiome composition, or in the mitigation of chronic inflammatory processes maintained by endotoxemia. Further in vitro and in vivo studies are needed before clinical use.

Effect of exogenous melatonin on the cellular response of Holstein heifer calves during vaccination.

Despite rigorous vaccination protocols, calf morbidity is the primary contributor to economic loss in the calf sector of the dairy industry. Melatonin has modulated immune response in other mature animal species. We hypothesized that exogenous melatonin may improve the cellular response to vaccination in dairy calves. Our objective was to evaluate the effect of exogenous melatonin on polymorphonuclear leukocyte (PMN) function in Holstein heifer calves during immunization. Sixty neonatal Holstein heifers were enrolled by birth cohort (block) and randomized to one of four treatments: control (CON), vaccination of 0.5mg ovalbumin on days 0 and 21 (VAC), implantation of 24mg melatonin on day 0 (MEL), or both melatonin and vaccine treatments (MVAC). Jugular blood was collected on days 0, 21, 42, and 63 to measure circulating melatonin, anti-ovalbumin immunoglobulin-G, and PMN function. Calves implanted with melatonin had greater circulating melatonin than non-implanted on day 21 (P < 0.01). Anti-ovalbumin IgG was greater for vaccinated than non-vaccinated calves (P < 0.01). Anti-ovalbumin IgG was greater for MVAC than VAC calves on day 63. Percent of cells and mean florescence intensity of cells performing oxidative burst decreased from day 0 to day 63 (P < 0.01) but were not affected by treatment (P ≥ 0.26). There was a tendency (P = 0.10) for an interaction of melatonin, vaccination, and day for the mean florescence intensity of cells performing phagocytosis where MVAC was greater than all other treatments on d 42. Exogenous melatonin may alter PMN function of calves during vaccination. Further research is needed to define the effect of melatonin on development of antigen-specific IgG during programmed vaccination protocols.

Effect of humic acid on oxidative stress and neuroprotection in traumatic spinal cord injury: an experimental study.

Traumatic spinal cord injury (TSCI) is an important health problem, especially in developing countries with additional socioeconomic loss. Humic acid (HA) usually has antioxidant, antiinflammatory, blood circulating, and antiviral effects. Hence, it was aimed herein to show the effect of HA on neuroprotection in a TSCI model. A TSCI model was used, in which 24 Wistar albino rats were divided into 4 groups: control group: subjected to only laminectomy; sham group: subjected to laminectomy + TSCI; HA 5 mg/kg group: subjected to laminectomy + TSCI + intraperitoneal (IP) injection of 5 mg/kg of HA; and HA 10 mg/kg group: subjected to laminectomy + TSCI + IP injection of 10 mg/kg of HA. Intracardiac blood samples were obtained preoperatively (preop), and at 1 and 24 h postoperatively (postop). The total antioxidant status (TAS), total oxidant status (TOS) and oxidative stress index (OSI) levels were evaluated in the serum. The motor functions were evaluated using the Modified Tarlov Score at 24 h postop. There were no significant changes in the TAS values between the sham and HA 5 mg/kg and HA 10 mg/kg groups (p = 0.77/0.21). However there was a significant decrease in the TOS values at 24 h postop when comparing the sham and HA 5 mg/kg groups (p = 0.02). The pathological evaluation showed a significant decrease in the severity of edema, hemorrhage, polymorphonuclear leucocyte (PNL) infiltration, and mononuclear leucocyte (MNL)/macrophage/microglia infiltration when compared with the control group (p < 0.05). There was a significant recovery at the paraplegia level when the HA 5 mg/kg and HA 10 mg/kg groups were compared with the control group (p < 0.001). The effects of HA in the early stages of TSCI on oxidative stress, histopathological changes, and neurological improvement were investigated herein. It is thought to be a potential therapeutic agent in acute TSCI but needs to be further evaluated to determine the extent of its effect on other neuroprotective pathways in larger series.