- Diphtheria- and tetanus-specific MBC detection by flow cytometry Protein biotinylation and formation of Ag-quantum dot (QD) complexes was performed using diphtheria toxin CRM197 (DT; List Biological Labs, CA), tetanus
- 132 Food allergy model. Peanut butter (3.75 mg; ~1 mg of protein; Kraft, Northfield, IL) with 10 µg 133 of cholera toxin (List Biologicals, Campbell, CA) in 0.5 mL of PBS was administered 134 intragastrically (Delvo SA, Biel, Switzerland) weekly for 4 weeks
Eosinophils have emerged as multifaceted cells that contribute to tissue homeostasis. However, the factors that control their frequency and function at mucosal sites remain unclear. Here, we investigated the role of the microbiota in regulating enteric eosinophils. We found that small intestinal (SI) eosinophilia was significantly greater in germ-free (GF) mice compared to specific pathogen free (SPF) controls. This phenomenon was associated with enteric overexpression of signals that mediate attraction, retention and survival of eosinophils, and was reversed by colonization. Additionally, we generated a novel strain of eosinophil-deficient GF mice. These mice displayed intestinal fibrosis and were less prone to allergic sensitization as compared to GF controls. Overall, our study demonstrates that commensal microbes regulate intestinal eosinophil frequency and function, which impacts tissue repair and allergic sensitization to food antigens. These data support a critical interplay between the commensal microbiota and intestinal eosinophils in shaping homeostatic, innate and adaptive immune processes in health and disease.
Anthrax toxin requires ZDHHC5-mediated palmitoylation of its surface-processing host enzymes
Sergeeva, OA;van der Goot, FG;
Proceedings Of The National Academy Of Sciences Of The United States Of America1161279-1288January 22, 2019
Product: Anti-Protective Antigen from B. anthracis (Goat)
... Primary antibodies used in this study that are commercially available include: rabbit anti-MEK2 (Santa Cruz Biotechnology sc-523, AB_2281672), goat antiprotective antigen from _Bacillus anthracis_ (List Biological Laboratories #771B), mouse anti-V5 (Thermo Fisher Scientific R960-25, AB_2556564), rabbit or goat anti-Furin (Thermo Fisher Scientific PA1-062, AB_2105077; R&D Systems AF1503), rabbit ... Primary antibodies used in this study that are commercially available include: rabbit anti-MEK2 (Santa Cruz Biotechnology sc-523, AB_2281672), goat antiprotective antigen from _Bacillus anthracis_ (List Biological Laboratories #771B), mouse anti-V5 (Thermo Fisher Scientific R960-25, AB_2556564), rabbit or goat anti-Furin (Thermo Fisher Scientific PA1-062, AB_2105077; R&D Systems AF1503), rabbit
For intoxication of cells, media was replaced over 105 752 cells previously seeded in a 12-well 753 plate. 31.7 nM PA alone (List Labs, #171E) or in the presence of 13.6nM LFN, LFN-ACD, or LFN754 RID was added to media and cells were incubated for 20 hr at 37°C in the presence of 5% CO2.
Multifunctional autoprocessing repeats-in-toxin (MARTX) toxins are pore-forming toxins that translocate multiple functionally independent effector domains into a target eukaryotic cell. Vibrio cholerae colonizes intestinal epithelial cells (IECs) and utilizes a MARTX toxin with three effector domains -- the actin cross-linking domain (ACD), the Rho inactivation domain (RID), and the alpha/beta hydrolase domain (ABH) -- to regulate innate immunity and enhance colonization. Whether these multiple catalytic enzymes delivered from a single toxin have coordinated function has not been explored. Using cultured IECs, we demonstrate ACD-induced cytoskeletal collapse activates a robust proinflammatory response that is blocked by the action of co-delivered RID and ABH. Thus, MARTX toxins utilize multiple enzymatic activities on a single toxin to silence the host response to both bacterial factors and effector function. Further, these data explain that V. cholerae utilizes the MARTX toxin to suppress intestinal inflammation and contribute to cholera being classically defined as non-inflammatory diarrheal disease.
Characterization of blood-brain barrier integrity in a B-cell-dependent mouse model of multiple sclerosis
Bell, L;Koeniger, T;Tacke, S;Kuerten, S;
Histochemistry And Cell BiologyJanuary 21, 2019
Product: Pertussis Toxin from B. pertussis, Lyophilized in Buffer
- For the induction of MP4-EAE, mice received 200 µg MP4 (Alexion Pharmaceuticals Inc) emulsified in CFA and 200 ng pertussis toxin (List Biological Laboratories, #181, Lot #18123611) in 500 µl sterile phosphate-buffered saline (PBS) on the day of immunization and
Recent studies with B-cell-depleting antibodies have demonstrated clinical success in the treatment of multiple sclerosis (MS) patients. While these antibodies efficiently target B cells in the blood, it is unclear how effective they are in the central nervous system (CNS), especially in the context of limited blood-brain barrier (BBB) permeability and the ongoing discussion on the relevance of B-cell aggregate formation in the brains of SP-MS patients. The aim of this study was to evaluate BBB integrity in the context of B-cell-dependent neuroinflammation in a mouse model of MS. C57BL/6 mice were actively immunized with either myelin oligodendrocyte glycoprotein peptide 35-55 to induce T-cell-dependent experimental autoimmune encephalomyelitis (EAE), or with the myelin basic protein-proteolipid protein fusion protein MP4 for additional B-cell dependence. BBB integrity was assessed using Evans Blue or fluorescein isothiocyanate-dextran injection, respectively, in combination with immunofluorescence staining for key components of the BBB. In both EAE models, tracer leakage into the CNS parenchyma was observed indicating BBB leakiness. Yet, intensity and distribution patterns of leakage differed between the two models. There was no difference in the severity of BBB damage comparing acute and chronic MP4-induced EAE, but the formation of B-cell aggregates was associated with local BBB impairment in this model. This study underscores that a leaky BBB is a characteristic feature of EAE, but it also suggests that extent and region specificity of BBB damage differs between individual EAE models that vary in the underlying immunopathology.