Optimized Protocol for Testing Multipurpose Contact Lens Solution Efficacy Against Acanthamoeba.

OBJECTIVES: To evaluate the interlaboratory and intralaboratory reproducibility of a proposed protocol for multipurpose contact lens solution (MPS) disinfection efficacy against Acanthamoeba. METHODS: Acanthamoeba castellanii and Acanthamoeba polyphaga and four MPS with different biocidal agents were used to evaluate the protocol in two different laboratories. In addition to the negative control, a positive control and neutralization control were used. One experiment was performed in triplicate, and all other experiments were performed in duplicate in each laboratory. Acanthamoeba trophozoites were grown axenically, and cysts were generated using the starvation method. Trophozoites and cysts at a concentration of 2.0 × 10 to 2.0 × 10 organisms per milliliter were exposed to the test MPS for 0, 4 or 6 (manufacturer's recommended soak time [MRST]), 8, and 24 hr. Survivors were determined by a limiting dilution method that used a most probable number evaluation. RESULTS: The positive and negative controls displayed consistent results and trends both within each laboratory and between each laboratory for trophozoites and cysts of both A. castellanii and A. polyphaga. The neutralization control consistently demonstrated the ability of the neutralizing agents to neutralize the MPS and the positive control and demonstrated no inhibition of Acanthamoeba by the negative control. Testing in triplicate and duplicate demonstrated the reproducibility of the protocol both within each laboratory and between the laboratories. Our results demonstrated that the MPS at the MRST and at 8 hr (likely overnight soak time) are generally more effective against trophozoites than they are against cysts. Only the MPS with hydrogen peroxide as the biocidal agent was able to provide a greater than three-log kill of cysts at the MRST and longer. Among the MPS we tested, trophozoites of A. castellanii and A. polyphaga showed similar responses. Some variability was observed when testing cysts of both species. In both laboratories, one nonhydrogen peroxide containing MPS had some effect (>1 log kill) on A. polyphaga cysts. This solution had no effect (<1 log kill) on A. castellanii cysts, A. castellanii trophozoites, and A. polyphaga trophozoites. CONCLUSIONS: The protocol that we have revised and evaluated is a well-controlled and reproducible procedure that can effectively evaluate the efficacy of MPS against Acanthamoeba trophozoites. Some variability was observed when testing the cyst stage.

Follicular dendritic cell regulation of CXCR4-mediated germinal center CD4 T cell migration.

Follicular dendritic cells (FDCs) up-regulate the chemokine receptor CXCR4 on CD4 T cells, and a major subpopulation of germinal center (GC) T cells (CD4(+)CD57(+)), which are adjacent to FDCs in vivo, expresses high levels of CXCR4. We therefore reasoned that GC T cells would actively migrate to stromal cell-derived factor-1 (CXCL12), the CXCR4 ligand, and tested this using Transwell migration assays with GC T cells and other CD4 T cells (CD57(-)) that expressed much lower levels of CXCR4. Unexpectedly, GC T cells were virtually nonresponsive to CXCL12, whereas CD57(-)CD4 T cells migrated efficiently despite reduced CXCR4 expression. In contrast, GC T cells efficiently migrated to B cell chemoattractant-1/CXCL13 and FDC supernatant, which contained CXCL13 produced by FDCs. Importantly, GC T cell nonresponsiveness to CXCL12 correlated with high ex vivo expression of regulator of G protein signaling (RGS), RGS13 and RGS16, mRNA and expression of protein in vivo. Furthermore, FDCs up-regulated both RGS13 and RGS16 mRNA expression in non-GC T cells, resulting in their impaired migration to CXCL12. Finally, GC T cells down-regulated RGS13 and RGS16 expression in the absence of FDCs and regained migratory competence to CXCL12. Although GC T cells express high levels of CXCR4, signaling through this receptor appears to be specifically inhibited by FDC-mediated expression of RGS13 and RGS16. Thus, FDCs appear to directly affect GC T cell migration within lymphoid follicles.