Female mice were immunized intravaginally with gonococcal outer membrane vesicles (OMV) plus microencapsulated IL-12, and challenged using an established model of genital infection with Neisseria gonorrhoeae. Whereas sham-immunized and control animals cleared the infection in 10–13 days, those immunized with OMV plus IL-12 cleared infection with homologous gonococcal strains in 6–9 days. Significant protection was also seen after challenge with antigenically distinct strains of N. gonorrhoeae, and protective anamnestic immunity persisted for at least 6 months after immunization. Serum and vaginal IgG and IgA antibodies were generated against antigens expressed by homologous and heterologous strains. Iliac lymph node CD4+ T cells secreted IFNγ, but not IL-4, in response to immunization, and produced IL-17 in response to challenge regardless of immunization. Antigens recognized by immunized mouse serum included several shared between gonococcal strains, including two identified by immunoproteomics approaches as EF-Tu and PotF3. Experiments with immunodeficient mice showed that protective immunity depended upon IFNγ and B cells, presumably to generate antibodies. The results demonstrated that immunity to gonococcal infection can be induced by immunization with a non-living gonococcal antigen, and suggest that efforts to develop a human vaccine should focus on strategies to generate Th1-driven immune responses in the genital tract.

Among retinoic acid receptors (RARs) alpha, beta, and gamma, the messenger RNA level of RAR-gamma is the most readily detectable by Northern blotting in human and mouse skin. This observation suggests that RAR-gamma may play a critical role in the modulation of the therapeutic benefits and side effects of retinoids in skin. To test this hypothesis, 11 RAR-gamma selective retinoids were synthesized based on three related structures. Each compound was found to prefer RAR-gamma when assessed by retinoid-induced transcriptional activity (RAR-gamma > RAR-beta > RAR-alpha). The apparent Kd for binding to recombinant receptor protein was found to follow a similar trend. To correlate this receptor selectivity with in vivo activity, the compounds were tested topically in the Rhino mouse utriculi reduction and rabbit irritation models, two assays widely used to screen retinoids for efficacy and side effects, respectively. The results indicated that for these compounds, both efficacy in the utriculi reduction assay and irritation potential in rabbits correlated positively with the RAR-gamma transactivation activity, with r2 of 0.9 and 0.8, respectively. These data suggest that RAR-gamma is an important regulator of retinoic acid efficacy in skin and further, that the irritation associated with the use of retinoids is most likely a receptor-mediated process.

The three retinoic acid receptors (RARΑ, RARΒ and RARΓ) are known to modulate the transcription of target genes through interaction of the individual receptors with their naturally occurring ligand, retinoic acid (RA). Since RA has multiple effects in vivo, considerable effort has recently been devoted to finding selective compounds to elucidate the functions of individual receptors and to relate these functions to specific in vivo effects. The racemic synthetic retinoid 6-[(5,5,8,8-tetramethyl- 5,6,7,8-tetrahydro-2 – naphthyl)hydroxy-methyl]-2-naphthalene carboxylic acid has recently been identified as an RARΓ-selective agonist. A synthetic method involving lipase-mediated transformation has been developed to prepare the individual enantiomers. Discrimination between the two enantiomers is seen in both transcriptional activity and binding to recombinant receptors with the (S)-enantiomer being the more active. Differences between the two compounds are also seen in the Rhino mouse utriculi reduction assay and the rabbit irritation model. In both animal models, the (S)-enantiomer consistently gave a greater response. Taken together, these results suggest that the activity and irritation seen with RA and related compounds is receptor mediated. Further, the strong selectivity of the compounds reported here for RARΓ suggests that this receptor plays an important role in these in vivo biological activities. The discrimination between these enantiomers may be useful in the design of novel retinoids with uniquely defined biological properties.

Genital infection with Neisseria gonorrhoeae (gonorrhea) is a significant cause of reproductive tract morbidity in women, leading to pelvic inflammatory disease, tubal factor infertility, and increased risk for ectopic pregnancy. WHO estimates that 78 million new infections occur annually worldwide. In the United States, >350,000 cases are reported annually, but the true incidence is probably >800,000 cases/year. Increasing resistance to currently available antibiotics raises concern that gonorrhea might become untreatable. Infection does not induce a state of immune protection against reinfection. Previous studies have shown that N. gonorrhoeae suppresses the development of adaptive immune responses by mechanisms dependent on the regulatory cytokines TGF-β and IL-10. This study shows that intravaginal treatment of gonococcal infection in female mice with microencapsulated IL-12 induces persisting anamnestic immunity against reinfection with N. gonorrhoeae, even of antigenically diverse strains, dependent on T-cell production of IFN-γ and B-cell production of antibodies.

The transcriptional response mediated by retinoic acid involves a complex series of events beginning with ligand recognition by a nuclear receptor. To dissect the amino acid contacts important for receptor-specific ligand recognition, a series of retinoic acid receptor (RAR) mutants were constructed. Transcriptional studies revealed that serine 232 (Ser 232 ) in RAR␣ and methionine 272 (Met 272 ) in RAR␥ are critical residues for the recognition of their respective receptor-selective analogs.The identification of these key amino acids in the ligand binding pocket is confirmed by the reported crystal structure of RAR␥. Interestingly, the serine at position 232 in RAR␣ gives an explanation for the observed differences in the affinity of the naturally occurring ligand, all-trans-retinoic acid (t-RA), in this receptor compared with that for the other receptors, since hydrogen bonding would not be permitted between the hydroxyl of serine and the hydrophobic linker of t-RA. Using this model, a molecular mechanism for the transcriptional antagonism of a synthetic analog is suggested that involves an alteration in the structure of the receptor protein in the region around the AF2 domain in helix 12.Retinoic acid and its analogs (retinoids) regulate cellular proliferation and differentiation in higher eukaryotes. The biological effects of these ligands are mediated by their binding to the retinoic acid receptors (RARs), 1 which are members of the superfamily of steroid-thyroid hormone nuclear receptors. These receptors act as transcriptional enhancers that bind in a sequence-specific manner to their response elements (retinoic acid response elements) located within the promoter region of distinct retinoid-responsive genes. RARs activate transcription of those genes after ligand bound to the receptor induces conformational changes leading to activation (1-4). A crystal structure of the ligand binding domain (LBD) of the closely related human retinoid-X receptor ␣ revealed a novel protein fold, an antiparallel ␣-helical sandwich, common to the members of this superfamily. Examination of this structure for a potential binding pocket for its ligand, 9-cis-retinoic acid, unveiled two large hydrophobic cavities within the N-terminal portion of the LBD in the vicinity of the s1,s2 ␤-hairpin and ␣-helix H5 (5). These findings are supported by results from ligand-photoaffinity binding experiments, which identified important residues for receptor-ligand binding in the LDBs of the glucocorticoid receptor (6) and RAR␣ (7,8). The amino acid sequence alignment of the nuclear receptor LBDs indicates that most of the residues identified by photoaffinity labeling or site-directed mutagenesis correspond to the 9-cis-retinoic acid receptor residues surrounding the putative 9-cis-retinoic acid binding pocket (9).Recently, the 2.0-Å structure of the LBD of human RAR␥ bound to all-trans-retinoic acid (t-RA) was reported showing details of ligand-receptor interactions (10). Several amino acids from ␣-helices H1, H3, H5, H11, H12, loops 6 -7 an...