Gang Liu

Cutaneous leishmaniasis (CL) is the most common clinical form of leishmaniasis, characterized by persistent skin ulcers and nodules. Standard chemotherapeutic agents have substantial toxicity and do nothing to repair the damaged tissue, an unmet need that motivates the search for adjunctive strategies. Mesenchymal stem cells (MSCs) can modulate macrophage activity and support tissue regeneration, yet their role in CL has received limited attention. In this study, we tested whether bone marrow-derived MSCs (BM-MSCs) could attenuate Leishmania mexicana-induced inflammation and facilitate skin repair. Indirect co-culture of BM-MSCs with infected RAW264.7 macrophages shifted the macrophage phenotype from M1 toward M2, with higher IL-10 and Arg-1 expression and lower iNOS and IL-1β. In BALB/c mice with established CL, three weekly intravenous injections of BM-MSCs reduced paw swelling, improved skin histology, decreased type I collagen deposition, lowered Integrin β1 and Cytokeratin 17 expression, and reduced tissue parasite load. Immunofluorescence confirmed a predominantly M2 macrophage distribution in treated lesions. We inferred that BM-MSCs acted on both the immune and reparative aspects of the disease process, supporting their potential as an adjunct to conventional anti-leishmanial therapy.

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Nocardia cyriacigeorgica causes bovine mastitis, reduces milk quantity and quality, and is often resistant to antimicrobials. Selenomethionine (SeMet) is a form of selenium, which reduces reactive oxygen species (ROS)-mediated apoptosis and intramammary infections. However, the protective effects of SeMet on N. cyriacigeorgica-infected bovine mammary epithelial cells (bMECs) are unclear. The objective of this study was to evaluate whether SeMet mitigated N. cyriacigeorgica-induced inflammatory injury, oxidative damage and apoptosis in bMECs. Cells were cultured with or without being pretreated with 40 µM of SeMet for 12 h, then challenged with N. cyriacigeorgica (multiplicity of infection = 5:1) for 6 h. Although N. cyriacigeorgica was resistant to lincomycin, erythromycin, enrofloxacin, penicillin, amoxicillin, cephalonium, cephalexin, and ceftriaxone, 40 μM SeMet increased cell viability and inhibited lactate dehydrogenase release in infected bMECs. Furthermore, N. cyriacigeorgica significantly induced mRNA production and protein expression of TNF-α, IL-1β, IL-6, and IL-8 at 6 h. Cell membrane rupture, cristae degeneration and mitochondria swelling were evident with transmission electron microscopy. Superoxide dismutase (SOD) and glutathione peroxidase (GSH-px) activities were down-regulated after 3, 6, or 12 h, whereas malondialdehyde (MDA) and ROS contents were significantly upregulated, with cell damage and apoptosis rapidly evident (the latter increased significantly in a time-dependent manner). In contrast, bMECs pretreated with 40 μM SeMet before infection, SOD, and GSH-px activities were upregulated (p < 0.05); MDA and ROS concentrations were downregulated (p < 0.05), and apoptosis was reduced (p < 0.05). In conclusion, 40 μM SeMet alleviated inflammation, oxidative stress and apoptosis induced by N. cyriacigeorgica in bMECs cultured in vitro.

Nocardia cyriacigeorgica causes bovine mastitis, reduces milk quantity and quality, and is often re-sistant to antimicrobials. Selenomethionine (SeMet) is a form of selenium, an essential trace el-ement. Although SeMet reduced ROS-mediated apoptosis and intramammary infections. How-ever, protective effects of SeMet on N. cyriacigeorgica-infected bovine mammary epithelial cells (bMECs) are unclear. The objective was to evaluate whether SeMet mitigated N. cyriacigeorgi-ca-induced inflammatory injury, oxidative damage and apoptosis in bMECs. Cells were cultured with or without being pretreated 40 µM of SeMet for 12 h, then challenged N. cyriacigeorgica (MOI=5:1) for 6 h. Although N. cyriacigeorgica was resistant to lincomycin, erythromycin, en-rofloxacin, penicillin, amoxicillin, cephalonium, cephalexin, and ceftriaxone, 40 μM SeMet in-creased cell viability and inhibited LDH release in infected bMECs. Furthermore, N. cyriacigeor-gica significantly induced mRNA production and protein expression of TNF-α, IL-1β, IL-6, and IL-8 at 6 h. Cell membrane rupture, cristae degeneration and mitochondria swelling were evi-dent with transmission electron microscopy. SOD and GSH-px activities were down regulated after 3, 6 or 12 h, whereas MDA and ROS contents were significantly upregulated, with cell damage and apoptosis rapidly evident (the latter increased significantly in a time-dependent manner). In contrast, bMECs pretreated with 40 μM SeMet before infection, SOD and GSH-px ac-tivities were upregulated (P < 0.05), MDA and ROS concentrations were downregulated (P < 0.05), and apoptosis was reduced (P < 0.05). In conclusion, 40 μM SeMet alleviated inflammation, oxi-dative stress and apoptosis induced by N. cyriacigeorgica in bMECs cultured in vitro.

Background: Leishmaniasis, caused byLeishmaniaspp. parasites, is an important zoonotic disease globally, posing severe threats to humans and animals. In the absence of effective vaccines, reliable serological diagnostic methods are critical for disease control. However, the enzyme-linked immunosorbent assay (ELISA) and immunochromatographic assay have limitations due to complexity, time required and/or sensitivity. Therefore, our objective was to develop an accurate, rapid and user-friendly detection method of canine leishmania antibody based on double-antigen sandwich homogeneous chemical luminescence.Methods: Homogeneous chemiluminescent technology was employed, and expressed recombinant fusion proteins containing full-length K9, K39 and K26 repeat sequences were used as diagnostic antigens. To establish a dual-antigen sandwich serological assay capable of detecting various antibody types, a factorial design was used to optimize concentrations of diagnostic antigen-receptor microspheres and of biotinylated diagnostic antigens, as well as of reaction solution composition and reaction duration. To evaluate and validate this newly developed method, we collected 41Leishmania-positive serum samples, 30Leishmania-negative control serum samples and 78 clinical serum samples for which no diagnostic information was available. Comparative analyses were performed using parasitological testing and an indirect ELISA as reference methods, focusing on diagnostic sensitivity and specificity.Results: Sodium dodecyl sulfate–polyacrylamide gel electrophoresis confirmed the purification of the diagnostic antigens, which exhibited clear bands without impurities. Based on results from the 41Leishmania-positive samples and 30Leishmania-negative samples, there was sufficient sensitivity to detect samples diluted up to 256-fold, with analytical specificity of 100%. Overall diagnostic sensitivity was 100% and diagnostic specificity was 93.3%. Diagnostic performance was highly consistent between the newly developed method and the indirect ELISA (Kappa = 0.82,P < 0.01). Testing could be completed within 35 min with the new methodConclusions: We have developed a novel double-antigen sandwich homogeneous chemical luminescence method to detect canineLeishmaniaantibodies, with high sensitively and specificity, a short incubation interval and a simple protocol. This streamlined approach not only offers a sensitive and efficient method for clinical diagnosis but also has great potential for use in automated testing.Graphical

Streptococcus uberis mastitis in cattle infects mammary epithelial cells. Although oxidative responses often remove intracellular microbes, S. uberis survives, but the mechanisms are not well understood. Herein, we aimed to elucidate antioxidative mechanisms during pathogenesis of S. uberis after isolation from clinical bovine mastitis milk samples. S. uberis’s in vitro pathomorphology, oxidative stress biological activities, transcription of antioxidative factors, inflammatory response cytokines, autophagosome and autophagy functions were evaluated, and in vivo S. uberis was injected into the fourth mammary gland nipple of each mouse to assess the infectiousness of S. uberis potential molecular mechanisms. The results showed that infection with S. uberis induced early oxidative stress and increased reactive oxygen species (ROS). However, over time, ROS concentrations decreased due to increased antioxidative activity, including total superoxide dismutase (T-SOD) and malondialdehyde (MDA) enzymes, plus transcription of antioxidative factors (Sirt1, Keap1, Nrf2, HO-1). Treatment with a ROS scavenger (N-acetyl cysteine, NAC) before infection with S. uberis reduced antioxidative responses and the inflammatory response, including the cytokines IL-6 and TNF-α, and the formation of the Atg5-LC3II/LC3I autophagosome. Synthesis of antioxidants determined autophagy functions, with Sirt1/Nrf2 activating autophagy in the presence of S. uberis. This study demonstrated the evasive mechanisms of S. uberis in mastitis, including suppressing inflammatory and ROS defenses by stimulating antioxidative pathways.

Giant pandas are the flagship species in world conservation, and include two subspecies, Ailuropoda melanoleuca qinlingensis (A. m. qinlingensis) and Ailuropoda melanoleuca sichuanensis (A. m. sichuanensis). Hematology and serum biochemistry studies are crucial to protecting giant pandas. Even though research on hematology and serum biochemistry are well-established in A. m. sichuanensis, research in A. m. qinlingensis is scarce. The study aimed to (1) establish a baseline for hemogram and reference intervals (RIs) for hematological and serum biochemical parameters in A. m. qinlingensis, (2) assess the possible variations in these parameters of A. m. qinlingensis based on age, gender, and storage condition of blood samples, and (3) compare the parameters to those of A. m. sichuanensis. Blood samples (n = 42) were collected from healthy A. m. qinlingensis (n = 21) housed in Shaanxi (Louguantai) Rare Wildlife Rescue and Breeding Research Center, and hematological (n = 25) and serum biochemical parameters (n = 18) were analyzed in March and December of 2019. The results showed no significant abnormality in the blood smears of all individuals in this study, except for a few serrated red blood cells, platelet aggregations, and occasionally giant platelets. Between sub-adult and adult A. m. qinlingensis, there were significant differences in five hematological and one serum biochemical parameter (p < 0.05), whereas six serum biochemical parameters were present when α = 0.1 (p < 0.1). Gender influenced % NEU, % LYM, % EOS, LYM, EOS, GGT, and CHOL of A. m. qinlingensis. The majority of the hematological and serum biochemical parameters of A. m. qinlingensis were different from those of A. m. sichuanensis regarding age and gender. The anticoagulant whole blood samples of A. m. qinlingensis stored at 2–8 °C for 24 h and the serum samples stored at −18 °C for 48 h had little influence on the values of hematological and serum biochemical parameters. In conclusion, this study provided a baseline of hemogram and established RIs for hematological and serum biochemical parameters of A. m. qinlingensis. RIs of A. m. sichuanensis reported before were not completely fit for A. m. qinlingensis, and age, gender, or the storage condition of blood samples influenced some of the parameters of A. m. qinlingensis. To the authors’ knowledge, this is the first report of a hemogram baseline and RIs for hematological and serum biochemical parameters of A. m. qinlingensis.

Bovine mastitis, the most prevalent and costly disease in dairy cows worldwide, decreases milk quality and quantity, and increases cow culling. However, involvement of microRNAs (miRNAs) in mastitis is not well characterized. The objective was to determine the role of microRNA-223 (miR-223) in regulation of the nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome and kelch like ECH-associated protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2) oxidative stress pathway in mastitis models induced by lipopolysaccharide (LPS) treatment of immortalized bovine mammary epithelial cells (bMECs) and murine mammary glands. In bMECs cultured in vitro, LPS-induced inflammation downregulated bta-miR-223; the latter interacted directly with the 3’ untranslated region (3’ UTR) of NLRP3 and Keap1. Overexpression of bta-miR-223 in bMECs decreased LPS and Adenosine 5’-triphosphate (ATP)-induced NLRP3 and its mediation of caspase 1 and IL-1β, and inhibited LPS-induced Keap1 and Nrf2 mediated oxidative stress, whereas inhibition of bta-miR-223 had opposite effects. In an in vivo murine model of LPS-induced mastitis, increased miR-223 mitigated pathology in the murine mammary gland, whereas decreased miR-223 increased inflammatory changes and oxidative stress. In conclusion, bta-miR-223 mitigated inflammation and oxidative injury by downregulating the NLRP3 inflammasome and Keap1/Nrf2 signaling pathway. This study implicated bta-miR-223 in regulation of inflammatory responses, with potential as a novel target for treating bovine mastitis and other diseases.