primary murine bmdms  (Jackson Laboratory)

Journal: mBio

Article Title: Multicomponent Gold-Linked Glycoconjugate Vaccine Elicits Antigen-Specific Humoral and Mixed T H 1-T H 17 Immunity, Correlated with Increased Protection against Burkholderia pseudomallei

doi: 10.1128/mBio.01227-21

Figure Lengend Snippet: Increased survival from AuNP-OpcP-LPS- or AuNP-OpcP1-LPS-immunized mice against a lethal intranasal challenge with B. pseudomallei K96243. (A) Graphical representation of the vaccination, challenge, and organ/tissue collection timeline. Two separate groups of C57BL/6 mice (at least n = 9) were immunized intranasally 3× in 2-week intervals with formulations containing 10 μg of protein, 10 μg of LPS, and 20 μg of CpG ODN 2395. In experiment one, a combination formulation (AuNP-Combo1-LPS) included equivalent amounts of protein (Hcp1, OmpW, OpcP, OpcP1, FlgL, and HA) from each candidate for a total of 10 μg of protein. A second animal experiment (at least n = 9) was conducted to evaluate histopathology, cell-mediated response, and BALF from vaccinated-only animals. (B) In experiment one, 3 weeks after the last vaccination, animals were challenged with 6 LD 50 (9.0 × 10 4 CFU per mouse) of B. pseudomallei K96243. (C to E) Lungs (C), livers (D), and spleens (E) of surviving animals were collected at 35 days postinfection to evaluate bacterial infection. Bacterial load was determined per gram of tissue, and representative panels for colonization are shown on log scale. All colonization data are shown as means ± standard errors of the means (SEM) of results determined per group. Statistical analyses were determined using the Kaplan-Meier method, followed by log rank test. Levels of significance compared to the adjuvant-only group: ***, P < 0.001; ****, P < 0.0001. ns, not significant.

Article Snippet: C57BL/6 murine bone marrow-derived primary macrophages (BMDM) (no. C57-6030; Cell Biologics Inc., Chicago, IL) were routinely grown in complete primary cell culture medium by following the manufacturer’s instructions (no. M3368; Cell Biologics).

Techniques: Histopathology, Infection

Journal: mBio

Article Title: Multicomponent Gold-Linked Glycoconjugate Vaccine Elicits Antigen-Specific Humoral and Mixed T H 1-T H 17 Immunity, Correlated with Increased Protection against Burkholderia pseudomallei

doi: 10.1128/mBio.01227-21

Figure Lengend Snippet: Intranasal immunization with optimized formulation (AuNP-Combo2-LPS) provides enhanced protection against inhalational melioidosis. C57BL/6 mice (at least n = 9 ) were immunized as described for Fig. 1 . The AuNP-Combo2-LPS vaccinated group contained equivalent amounts of protein (OpcP and Opcp1) from each candidate for a total of 10 μg of protein. (A to D) After intranasal challenge with 5 LD 50 (7.5 × 10 4 CFU per mouse) of B. pseudomallei K96243 (A), the lungs (B), livers (C), and spleens (D) of surviving animals were collected at 35 days postinfection, and bacterial enumeration was performed. Bacterial load was determined per gram of tissue, and representative panels for colonization are shown on a log scale. (E) Histopathological analysis from lungs, livers, and spleens of a representative mouse from each surviving group. Lung sections from the AuNP-OpcP-LPS vaccination group showed increased pathological findings compared to the AuNP-OpcP1-LPS and AuNP-Combo2-LPS groups. The liver sections from the AuNP-OpcP-LPS immunization group showed the most inflammation and evidence of liver injury. Spleen sections from surviving animals all showed similar histopathological findings, with the most pronounced in the AuNP-OpcP-LPS vaccine group. At the endpoint of the experiment, tissues were harvested from three mice of the 10 survivors. The tissues were fixed, sectioned, and stained with hematoxylin and eosin. Images are representative of three mice. Scale bar, 500 μm. All colonization data are shown as means ± standard errors of the means (SEM) of results determined per group. Statistical analyses were determined using the Kaplan-Meier method, followed by log rank test. Levels of significance compared to the adjuvant-only group: ***, P < 0.0005; ****, P < 0.0001. ns, not significant.

Article Snippet: C57BL/6 murine bone marrow-derived primary macrophages (BMDM) (no. C57-6030; Cell Biologics Inc., Chicago, IL) were routinely grown in complete primary cell culture medium by following the manufacturer’s instructions (no. M3368; Cell Biologics).

Techniques: Staining