Interestingly 39% of the suspected cases had been vaccinated against diphtheria, indicating that there is a need to improve the current vaccination strategy and/or the vaccine. component of multivalent tetanus/ diphtheria vaccine in young and old mice. This information could be useful for the future design of vaccines for the elderly. Keywords: Diphtheria, Tetanus, Vaccination, GM-CSF, Antibody 1.?Introduction Immunization is a powerful weapon for the prevention Foxo1 of infectious diseases, such as smallpox, tetanus or diphtheria. Diphtheria vaccines belong to the most frequently applied vaccines worldwide, with a long history dating back to the 1920s, when Glenny and Hopkins inactivated the diphtheria toxin and showed a high immunity index of the toxoid [1]. In the early 1930s the diphtheria toxoid was found to be more immunogenic when adsorbed to aluminum salts as carrier [2]. To date, no drastic changes have been made to the vaccine. The toxoid is formulated with aluminum-hydroxide as an adjuvant in multivalent vaccines combined with Enfuvirtide Acetate(T-20) other antigens, such as pertussis, polio and the most prevalent, tetanus toxoid. Our group has a long-standing history studying tetanus and diphtheria vaccinations in persons of different ages and we have repeatedly shown good protection against tetanus, while protection against diphtheria was insufficient [3C5]. Studies by other groups have also demonstrated insufficient protection against diphtheria Enfuvirtide Acetate(T-20) in adults [6C8]. Therefore we were curious how one could improve the immune responsiveness to the diphtheria component of the vaccine, which is of particular importance for elderly persons who are unprotected against diphtheria, more frequently than young [5,9]. Increasing the diphtheria dosage is not a viable option due to reported side effects of booster vaccines containing high diphtheria toxoid concentrations [10,11]. In a recent study we found a positive correlation between diphtheria-specific granulocyte macrophage-colony stimulating factor (GM-CSF) production by CD4+ T cells, and Enfuvirtide Acetate(T-20) peripheral diphtheria-specific antibodies in adults [5]. GM-CSF is a cytokine mainly produced by activated leukocytes and recognized, via the GM-CSF receptor, by granulocytes, monocytes, macrophages, as well as DCs and their precursors [12,13]. GM-CSF is known to stimulate chemotaxis, proliferation and differentiation, and is also generally recognized as a pro-inflammatory cytokine [14,12,15]. There are numerous studies that successfully tested GM-CSF as an adjuvant for vaccines, thereby improving immune responses to H5N1 influenza virus, a crude leishmania antigen vaccine, hepatitis B virus, human immunodeficiency virus (HIV), effect of GM-CSF on immune responses to diphtheria and tetanus immunization in young and aged mice using multivalent vaccines containing diphtheria toxoid, tetanus toxoid, acellular pertussis, and inactivated polio virus, to model the human immunization schedule. 2.?Materials and methods 2.1. Animals Eight week and 17 month old C57BL/6JRj male mice were purchased from JANVIER LABS (Le Genest-Saint-Isle, France). Mice were provided standard food and water (25 g pertussis toxoid (PT), 25 g pertussis filamentous haemag-glutinin (FHA), and 8 g pertactin), and three types of inactivated polio viruses (40 D-antigen units type 1: Mahoney strain; 8 D-antigen units type 2: MEF-1 strain; 32 D-antigen units type 3: Saukett strain; GlaxoSmithKline, London, United Kingdom) per single human dose (0.5 mL), or 30 L of diluted Boostrix Polio? containing per single human dose (0.5 mL) not less than 2 IU diphtheria toxoid, and not less than 20 IU tetanus toxoid, three purified antigens of (8 g PT, 8 g FHA and 2.5 g pertactin) and the same three types of inactivated polio viruses as included in Infanrix? IPV at the same dosages (GlaxoSmithKline) of the right or left leg. For all vaccinations, Infanrix? IPV from the same batch was used. The same was true for Boostrix Polio?. Both vaccines were diluted 1:2.4 with Enfuvirtide Acetate(T-20) phosphate buffered saline (PBS; Merck KGaA, Darmstadt, HE, Germany). GM-CSF-treated mice received 100 ng of recombinant mouse GM-CSF (Biolegend, San Diego, CA, USA) diluted in 20 L PBS with 0.2% Bovine Serum Albumin (BSA Fraction V; GE Healthcare, Little.