Brucellosis is a prevalent zoonotic disease that poses a serious threat to human health, particularly during pregnancy, where infection is strongly associated with spontaneous abortion. However, the mechanisms by which Brucella infection disrupts placental function remain poorly understood. Trophoblast cells are essential for placental development and represent primary targets of Brucella infection at the maternal-fetal interface. In this study, we investigated the effects of Brucella outer membrane lipoprotein 16 (L16) on trophoblast cell fate using the human JEG-3 cell line. L16 markedly suppressed trophoblast cell proliferation and induced membrane damage, as evidenced by increased lactate dehydrogenase release. Unlike lipopolysaccharide stimulation, L16 triggered a lytic, non-apoptotic form of programmed cell death characterized by plasma membrane ballooning. Mechanistically, L16 activated caspase-3 and promoted cleavage of gasdermin E (GSDME) into its pore-forming N-terminal fragment, thereby executing GSDME-dependent pyroptosis. Silencing of caspase-3 abolished GSDME cleavage and significantly attenuated pyroptotic cell death, confirming the essential role of the caspase-3/GSDME axis. Further analysis revealed that L16 reprogrammed inflammatory and survival signaling in trophoblast cells. L16 suppressed interleukin-6 (IL-6) trans-signaling by downregulating IL-6 receptor expression and up-regulating suppressor of cytokine signaling 3 (SOCS3), leading to inhibition of the JAK2/STAT3 pathway. Concurrently, L16 inhibited PI3K/AKT signaling and reduced phosphorylation of X-linked inhibitor of apoptosis protein (XIAP), thereby releasing inhibitory constraints on caspase-3 activation. Transcriptomic profiling supported coordinated repression of IL-6/JAK/STAT3 and PI3K/AKT pathways following L16 exposure. Collectively, these findings identify L16 as a key Brucella virulence factor that induces trophoblast pyroptosis by rewiring IL-6 trans-signaling and survival pathways, providing new mechanistic insight into Brucella-associated pregnancy failure.

Background:In fetal and neonatal alloimmune thrombocytopenia (FNAIT), maternal antibodies react with alloantigen expressed on fetal platelets, leading to their clearance via antibody-dependent phagocytosis. In Caucasians, most FNAIT cases are caused by anti-HPA-1a antibodies. In contrast, anti-HLA class I antibodies are rarely found in FNAIT, but are frequently implicated in cases of platelet transfusion refractoriness (PTR). This phenomenon leads to ongoing debate regarding the role of anti-HLA class I antibodies in FNAIT. In this study, we investigated the platelet clearance mediated by anti-HLA class I antibodies in whole blood both in vitro and in vivo. Methods:Clearance of opsonized platelet was analyzed by platelet phagocytosis assay and by antibody administration to Balb/c female mice. Results:To mimic FNAIT conditions, whole blood was pretreated with anti-HLA antibodies before the phagocytosis of anti-HPA-1a antibody-opsonized platelets. Compared to untreated whole blood, anti-HLA-ABC and anti-HLA-DR IgG antibodies inhibited the phagocytosis of anti-HPA-1a-antibody-opsonized platelets. Similar results were obtained with purified monocytes, indicating that anti-HLA-ABC antibodies bound to monocytes can interfere with antibody-mediated platelet phagocytosis. Furthermore, the administration of anti-MHC-I antibodies to mice led to a significant decrease in the platelet count within 24 h. However, anti-αIIbβ3 antibody administration resulted in significantly higher platelet clearance over different time points. Analysis of antibody-bound platelets showed the presence of anti-αIIbβ3 antibodies on the platelet surface, but not on monocytes. In contrast, anti-MHC-I antibodies were found on both platelets and monocytes. Interestingly, monocytes exhibited higher levels of anti-MHC-I binding than platelets (87.0% vs. 25.5%), most likely because platelets express significantly fewer HLA class I antigens than monocytes, as indicated by our flow cytometric analysis of whole blood. Conclusions:These results indicated that anti-MHC-I antibodies preferentially bind to monocytes rather than platelets in whole blood and can be cleared by monocytes via endocytosis. Furthermore, we found that the presence of anti-HLA class I antibodies did not significantly influence platelet clearance induced by anti-αIIbβ3 antibodies. The question of whether these observations can explain the controversial opinions regarding the relative roles of anti-HLA class I and anti-αIIbβ3 antibodies in FNAIT requires further assessment in a murine model of FNAIT.

BACKGROUND:This study sought to examine the suppression of the NK4 (which is a fragment that originates from the trypsin digestion of the hepatocyte growth factor) gene as mediated by new nano material polyamidoamine (PAMAM) dendrimers in the growth of breast cancer cells MDA-MB-231 and MCF-7, and the therapeutic effects in a nude mice model of transplanted tumor cell MDA-MB-231.METHODS:We built PAMAM-NK4 nano particles and detected the in vitro transfection rate. Nano complexes and blank plasmid PAMAM dendrimers were transfected to MDA-MB-231 and MCF-7 cells, respectively. The western-blotting method, MTT experiment method, and bead method were used to detect the effects of the nano complexes on NK4 protein expression, cell proliferation, and cell apoptosis. The nude mice model of transplanted tumor cell MDA-MB-231 comprised 40 nude female mice who were subject to injections. The mice were randomly divided into four groups, comprising 10 mice per group. The control, blank plasmid and treatment groups were subcutaneously injected with 0.2 mL of 0.9% NaCl (Sodium chloride) solution, 0.2 mL of plasmid solution (including 100 µg PAMAM pcDNA3.1(-) blank plasmid nano complexes) and 0.2 mL of plasmid solution (including PAMAM-NK4 100 µg) beside the tumor inoculation spot, respectively. The positive control group was intraperitoneally injected with 0.2 mL of doxorubicin solution, including 100 µg doxorubicin. Western blotting was used to detect the NK4 protein expression of the transplanted tumor tissues of the various groups.RESULTS:NK4 protein was successfully expressed in MDA-MB-231 and MCF-7 cells transfected with PAMAM-NK4 nano particles, and cell proliferation was suppressed and cell apoptosis was induced. The tumor volumes and masses of the treatment and positive control groups were obviously smaller than those of the control group. The differences were statistically significant (P<0.05). The treatment group had an obviously higher mean value of NK4 protein expression than the control group. The differences were statistically significant (P<0.05).CONCLUSIONS:PAMAM-NK4 nano complexes suppress the growth of the breast cancer cells MDA-MB-231 and MCF-7, and had a treatment effect on this tumor nude mice model of breast cancer cells.

Background Brucella species are Gram-negative intracellular bacteria that causes severe inflammatory diseases in animals and humans. Two major lipoproteins (L19 and L16) of Brucella outer membrane proteins were studied to explore the association with inflammatory response of human monocytes (THP-1). Methods Activated THP-1 cells induced with recombinant L19 and L16 were analyzed in comparison with unlipidated forms (U19 and U16) and lipopolysaccharide (LPS) of Brucella melitensis, respectively. Results Secretion of inflammatory factors tumor necrosis factor-α, interleukin (IL)-6, and IL-1β was significantly increased from L19, L16, or both stimulated THP-1 cells. High secretion of IL-18 was detected only from L19-induced cells. Signaling of those cytokine responses was identified mainly through the P38-mitogen-activated protein kinase pathway, and signaling of L19-induced IL-1β response partly occurred via necrosis factor-κB. While exploring different forms of IL-18, we found that L19-induced production of active IL-18 (18 kD) occurred through upregulating NLRP3 and activating caspase-1, whereas L16-induced production of inactive IL-18 fragments (15 kD and 16 kD) occurred through activating caspase-8/3. We also found that L19 upregulated phosphorylation of XIAP for inhibiting caspase-3 activity to cleave IL-18, whereas L16 activated caspase-3 for producing GSDME-N and leading to pyroptosis of THP-1 cells. Conclusions Brucella L19 and L16 differentially induce IL-18 response or pyroptosis in THP-1 cells, respectively.

Brucellosis is a serious zoonosis occurring mainly in developing countries, and its diagnosis is largely dependent on serologic detection and bacterial culture. In this study, we developed the murine monoclonal antibodies (mAbs) against a conserved and major outer membrane protein 25 (Omp25) of Brucella species (B. spp.) for use in clinical diagnosis. The mAbs to Omp25 were produced by hybridoma technique, which were utilized for developing various immunoassays for detection of Brucellae, including Western blot (WB), enzyme-linked immunosorbent assay (ELISA), immunochemical staining (ICS), immunofluorescence staining (IFS), and flow cytometry assay (FCM). A number of five mAbs (2B10, 4A12, 4F10, 6C12, and 8F3) specific to Omp25 were selected, including 2 IgG1, 2 IgG2a, and 1 IgG2b. Among them, mAbs 6C12, 8F3, and 4A12 reacted highly with B. melitensis (M5-90), B. abortus (S19, 104M, and 2308), and B. suis strain (S2). No cross-reactivity with Yersinia enterocolitica O:9, Salmonella spp., and Escherichia coli was found. By mapping Omp25 epitopes, mAb 6C12 was found as reacting with a semi-conformational epitope, and mAbs 4A12 and 8F3 as recognizing a different linear epitope, respectively. The paired mAbs were tested for detecting Brucella species, suggesting that 8F3 was suitable for solid phase capture and 6C12 or 4A12 was suitable for conjugation with HRP for detection of Brucella Omp25 in ELISA. The FCM was established by mAb 6C12 for detecting intracellular Brucellae-infected peripheral blood mononuclear cells (PBMCs) from brucellosis patients. In conclusion, mAbs against Omp25 are precious reagents for detection of Brucellae in clinical samples with various immunoassays. mAb 6C12-based FCM could be potentially used for the monitoring of therapeutic efficacy for brucellosis in clinical practice.