RESUMEN
Currently, antimicrobial peptides have attracted considerable attention because of their broad-sprectum activity and low prognostic to induce antibiotic resistance. In our study, for the first time, a series of side-chain hybrid dimer peptides J-AA (Anoplin-Anoplin), J-RR (RW-RW), and J-AR (Anoplin-RW) based on the wasp peptide Anoplin and the arginine- and tryptophan-rich hexapeptide RW were designed and synthesized by click chemistry, with the intent to improve the antimicrobial efficacy of peptides against bacterial pathogens. The results showed that all dimer analogues exhibited up to a 4-16 fold increase in antimicrobial activity compared to the parental peptides against bacterial strains. Furthermore, the antimicrobial activity was confirmed by time-killing kinetics assay with two strains which showed that these dimer analogues at 1, 2×MIC were rapidly bactericidal and reduced the initial inoculum significantly during the first 2-6h. Notably, dimer peptides showed synergy and additivity effects when used in combination with conventional antibiotics rifampin or penicillin respectively against the multidrug-resistant strains. In the Escherichia coli-infected mouse model, all of hybrid dimer analogues had significantly lower degree of bacterial load than the untreated control group when injected once i.p. at 5mg/kg. In addition, the infected mice by methicillin-resistant (MRSA) strain could be effectively treated with J-RR. All of dimer analogues had membrane-active action mode. And the membrane-dependent mode of action signifies that peptides functions freely and without regard to conventional resistant mechanisms. Circular dichroism analyses of all dimer analogues showed a general predominance of α-helix conformation in 50% trifluoroethanol (TFE). Additionally, the acute toxicities study indicated that J-RR or J-AR did not show the signs of toxicity when adult mice exposed to concentration up to 120mg/kg. The 50% lethal dose (LD50) of J-AA was 53.6mg/kg. In conclusion, to design and synthesize side chain-hybrid dimer analogues via click chemistry may offer a new strategy for antibacterial therapeutic option.
Asunto(s)
Antiinfecciosos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Escherichia coli/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Venenos de Avispas/farmacología , Secuencia de Aminoácidos/genética , Animales , Antibacterianos/química , Antiinfecciosos/química , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/genética , Sinergismo Farmacológico , Escherichia coli/patogenicidad , Cadenas J de Inmunoglobulina/química , Cadenas J de Inmunoglobulina/farmacología , Staphylococcus aureus Resistente a Meticilina/patogenicidad , Ratones , Pruebas de Sensibilidad Microbiana , Multimerización de Proteína , Rifampin/farmacología , Triazoles/química , Venenos de Avispas/química , Venenos de Avispas/genética , Avispas/químicaRESUMEN
As in other mammals, immunoglobulin A (IgA) in the horse has a key role in immune defense. To better dissect equine IgA function, we isolated complementary DNA (cDNA) clones for equine J chain and polymeric Ig receptor (pIgR). When coexpressed with equine IgA, equine J chain promoted efficient IgA polymerization. A truncated version of equine pIgR, equivalent to secretory component, bound with nanomolar affinity to recombinant equine and human dimeric IgA but not with monomeric IgA from either species. Searches of the equine genome localized equine J chain and pIgR to chromosomes 3 and 5, respectively, with J chain and pIgR coding sequence distributed across 4 and 11 exons, respectively. Comparisons of transcriptional regulatory sequences suggest that horse and human pIgR expression is controlled through common regulatory mechanisms that are less conserved in rodents. These studies pave the way for full dissection of equine IgA function and open up possibilities for immune-based treatment of equine diseases.
Asunto(s)
Inmunoglobulina A/metabolismo , Cadenas J de Inmunoglobulina/farmacología , Proteínas Recombinantes/metabolismo , Secuencia de Aminoácidos , Animales , Afinidad de Anticuerpos , Cromosomas de los Mamíferos/genética , Clonación Molecular , Reacciones Cruzadas/inmunología , Caballos/inmunología , Humanos , Inmunoglobulina A/genética , Inmunoglobulina A/inmunología , Cadenas J de Inmunoglobulina/genética , Cadenas J de Inmunoglobulina/inmunología , Datos de Secuencia Molecular , Unión Proteica , Multimerización de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Especificidad de la EspecieRESUMEN
The aim of this study was to determine the effect of expressing a recombinant anti-Kell immunoglobulin (Ig) M from two cell lines, CH0 and NS0, on its ability to function as a diagnostic antibody. As a polymeric immunoglobulin, IgM is able to directly agglutinate red blood cells (RBCs), making it a useful blood grouping reagent. To simplify expression, recombinant human IgM (rIgM) from NS0 (a mouse myeloma line) and CHO (Chinese hamster ovary line) cells was expressed in the absence of human J chain. Whereas NS0 expresses mouse J chain, rIgM expressed from CH0 cells lack J chain. Although the ability to polymerize resides within the tailpiece of IgM heavy chain, J chain can influence the polymeric state. This in turn could affect the ability of rIgM to bind its antigen. The variable region of the heavy chain of an anti-Kell IgG was grafted onto the constant region of human IgM and co-expressed with light chain derived from the same antibody. rIgM was purified from each cell line and the strength of direct agglutination assessed. Both cell lines produced polymeric rIgM that was able to specifically bind the target antigen and to directly agglutinate RBCs to the same degree. The presence or absence of J chain did not affect the ability of the rIgM to bind the Kell antigen or the strength of agglutination. The presence of J chain is not required for the production of a functional rIgM for use as a diagnostic reagent. CHO and NS0 lines are both suitable for production of such a reagent.
Asunto(s)
Tipificación y Pruebas Cruzadas Sanguíneas/métodos , Cadenas J de Inmunoglobulina/farmacología , Inmunoglobulina M/inmunología , Sistema del Grupo Sanguíneo de Kell/inmunología , Animales , Anticuerpos , Línea Celular , Humanos , Proteínas RecombinantesRESUMEN
We evaluated the efficacy of the Ligand Epitope Antigen Presentation System (L.E.A.P.S.trade mark) in preventing or treating experimental autoimmune myocarditis (EAM) in A/J mice. L.E.A.P.S. (here, J-My-1) is a conjugate of the myocarditogenic peptide of cardiac myosin MyHCalpha(334-352) (My-1) and J peptide, derived from the sequence of human beta-2 microglobulin. Remarkably, early prophylactic (J-My-1 injected on days -14 and -7 before EAM induction), late prophylactic (J-My-1 injected on days 0, 7, 14, and 21), and therapeutic (J-My-1 injected on days 7, 14, and 21 or 10, 17 and 24) administration of J-My-1 significantly decreased the incidence and severity of EAM. However, extended therapeutic treatment was associated with anaphylaxis and death, corresponding with global immune activation associated with J-My-1 treatment. In J-My1-treated animals, we observed expanded numbers of activated CD69+ and CD44+ CD4+ and CD8+ T cells in the spleens. J-My-1 treatment also increased the proportion of CD11c+ dendritic cells in spleens and induced strong production of anti-J-My-1 specific antibodies. J-My-1 injections resulted in decreased levels of chemokines MIP-1alpha and IP-10 in hearts. We propose that J-My-1 treatment interferes with trafficking of autoaggressive immune cells to the heart.