Comparative Analysis of the Osmoprotective Effects of Daily Disposable Contact Lens Packaging Solutions on Human Corneal Epithelial Cells.

Purpose: Contact lens (CL) wear challenges the balance of the ocular surface environment by increasing water evaporation and tear osmolarity. Maintaining ocular surface homeostasis during CL wear remains a goal of lens manufacturers and an important consideration for eye care professionals. The purpose of this study was to measure the metabolic activity and inflammatory responses of a transformed human corneal epithelial cell (THCEpiC) line under hyperosmotic conditions in the presence of CL packaging solutions. Methods: CL packaging solutions sampled from seven daily disposable silicone hydrogel CL blister packages were prepared at 25% and made hyperosmolar (400 mOsm/kg) with NaCl. THCEpiCs were incubated with each solution for 24 hr, after which cell culture supernatants were collected. THCEpiC metabolic activity was determined by an alamarBlue assay. Concentrations in cell culture supernatants of inflammatory cytokine (interleukin [IL]-6) and chemokine (IL-8), as well as monocyte chemoattractant protein-1 (MCP-1), were quantitated by specific enzyme-linked immunosorbent assays. Results: THCEpiC metabolic activity under hyperosmolar conditions decreased in the presence of somofilcon A and senofilcon A solutions (p=0.04 and 0.004, respectively), but no other solution (all p≥0.09). Concentrations of IL-6 increased in the presence of delefilcon A, somofilcon A, narafilcon A, and senofilcon A solutions (all p≤0.001), but no other solution (all p≥0.08), while those of IL-8 increased in the presence of all solutions (all p≤0.03) but kalifilcon A (p>0.99), and those of MCP-1 increased in the presence of delefilcon A, verofilcon A, somofilcon A, and stenfilcon A solutions (all p<0.0001), but no other solution (all p>0.99). Conclusion: CL packaging solutions differ in their capacity to inhibit epithelial inflammation. THCEpiC inflammatory response was less in the presence of a CL packaging solution containing osmoprotectants than in solutions lacking osmoprotectants under moderately hyperosmolar conditions in vitro. Clinical studies are warranted to further substantiate the benefit of osmoprotectants.

Effect of Contact Lens Solutions in Stabilizing the Activity of Tear Lysozyme.

Purpose: Interactions between tear proteins and the interfaces of contact lenses can be complex and can influence contact lens wear success. Tear proteins, including lysozyme, function to maintain the balance of ocular surface homeostasis, as evidenced by the effects of its conformation relative to stabilizing the tear film and its potential impact on corneal epithelial cells. Contact lens manufacturers include components in lens care and blister package solutions to help stabilize the tear film and preserve homeostasis. This in vitro study was performed to evaluate the ability of daily disposable contact lens package solutions to stabilize lysozyme and preserve its native conformation under denaturing conditions. Methods: Lysozyme was added to contact lens solutions sampled from kalifilcon A, etafilcon A, senofilcon A, narafilcon A, nelfilcon A, verofilcon A, delefilcon A, somofilcon A, and stenfilcon A blister packages, then mixed with the protein denaturant sodium lauryl sulfate. Lysozyme activity was evaluated by adding test solutions to a suspension of Micrococcus luteus. Native lysozyme lyses the Micrococcus luteus cell wall, which decreases suspension turbidity. Stabilization of lysozyme activity was determined by comparing suspension turbidity before and after exposure to test solutions. Results: Lysozyme stabilization was 90.7% for kalifilcon A solution, a statistically significant improvement (p < 0.05) compared to phosphate buffered saline (PBS, negative control). No significant improvement was observed with any other contact lens solution (all lysozyme stabilization < 5.00%). Conclusion: The representative tear protein lysozyme was significantly more stable in the novel kalifilcon A contact lens solution containing multiple moisturizers and osmoprotectants than in PBS or other daily disposable contact lens solutions. The lysozyme activity assay provides mechanistic evidence that the kalifilcon A contact lens solution can stabilize proteins under conditions that typically denature proteins, which may contribute to maintaining ocular surface homeostasis.

Increased concentration of hyaluronan in tears after soaking contact lenses in Biotrue multipurpose solution.

PURPOSE: This study was conducted to determine 1) the concentration of hyaluronan (HA) in the tear films of contact lens (CL) wearers versus non-CL wearers and 2) whether HA sorbed from Biotrue, an HA-containing multipurpose solution (MPS), onto senofilcon A lenses affects the concentration of HA in tears after 2 hours of wear. PATIENTS AND METHODS: Tears of habitual CL wearers and non-CL wearers were collected on Schirmer strips at baseline and after 2 hours of wear of senofilcon A CLs that had first been either rinsed with Sensitive Eyes Saline or soaked in Biotrue MPS for 14 hours. HA concentrations were measured by enzyme-linked immunosorbent assay (ELISA) and adjusted for sample volumes. RESULTS: No difference in baseline concentrations of HA in tears was found between CL wearers and non-CL wearers (P=0.07), nor between males and females (P=0.06). However, age was significantly negatively associated with HA concentration (P<0.01), and mostly, CL wear contributed to a significant association (P<0.01). Among saline-rinsed CL wearers, no change in HA concentration in tears was observed after 2 hours of wear (P=0.38). By contrast, a significant increase in HA concentration was observed in the tears from eyes that had worn CLs soaked in Biotrue MPS when compared to baseline (P=0.01) or to saline-rinsed control (P=0.03). CONCLUSION: 1) In this study population, no difference in baseline concentration of HA was observed between CL wearers and non-CL wearers, and 2) after 2 hours of wear of senofilcon A lenses that were soaked in Biotrue MPS, HA concentrations in the tear films of CL wearers increased.

Reduction of nasal colonization of nontypeable Haemophilus influenzae following intranasal immunization with rLP4/rLP6/UspA2 proteins combined with aqueous formulation of RC529.

Nontypeable Haemophilus influenzae (NTHi) and Moraxella catarrhalis are common causative agents of human mucosal infections. To formulate a mucosal vaccine against these pathogens, recombinant lipidated P4 (rLP4) and P6 (rLP6) proteins of NTHi and ubiquitous cell surface protein A (UspA) of M. catarrhalis were used for active immunization experiments in a mouse nasal challenge model. BALB/c mice were immunized intranasally with these proteins formulated with a chemically synthesized adjuvant, RC529 in an aqueous formulation (RC529-AF). Three weeks after the last immunization, these animals were challenged intranasally with NTHi strain SR7332.P1 and nasal colonization measured 3 days later. To determine local and systemic immune responses, bronchoalveolar washes (BAW) and sera were collected prior to NTHi challenge. The serum and mucosal samples were analyzed by ELISA for rLP4, rLP6 and UspA2 protein-specific IgG, IgG subclass and IgA antibody titers and bactericidal titers were determined against the TTA24 and 430-345 strains of M. catarrhalis. Results of these experiments show that these proteins combined with RC529-AF administered intranasally to mice elicited (1) significantly increased rLP4/rLP6/UspA2 protein-specific circulating IgG and IgA antibody responses; (2) local rLP4/rLP6/UspA2-specific IgA responses in the respiratory tract; and (3) more than a two log reduction of nasal colonization of NTHi strain SR7332 from the nasal tissues of mice. The serum IgG subclass distribution was predominantly IgG2a, representing a Th1 response. The antiserum also exhibited bactericidal activities to several strains of M. catarrhalis. These data indicate that intranasal immunization with rLP4/rLP6/UspA2 proteins combined with RC529-AF may be able to provide a way for inducing local mucosal immunity and for prevention of otitis media in children.

Inhibition of respiratory syncytial virus infection with the CC chemokine RANTES (CCL5).

Respiratory syncytial virus (RSV) is a major cause of respiratory tract disease in infants, aged adults, and immunosuppressed patients. The only approved medicines for RSV disease are administration of prophylatic antibodies or treatment with a synthetic nucleoside. Both approaches are expensive and the latter is not without risk and of controversial benefit. The present investigation studied whether pharmaceutical or biologic compounds based upon chemokines might be useful in preventing RSV disease. Of interest was RANTES/CCL5, which inhibits infection by HIV strains that use chemokine receptor (CCR)-5 as co-receptor. Herein, we report that prior or simultaneous treatment of HEp-2 cells with recombinant human CCL5 provides dose-dependent inhibition of infection with RSV. Other recombinant chemokines (MIP-1alpha/CCL3, MIP-1beta/CCL4, MCP-2/CCL8, eotaxin/CCL11, MIP-1delta/CCL15, stromal cell derived factor (SDF)-1alpha/CXCL12) were not inhibitory. The data suggested that CCL5 might inhibit infection by blocking fusion (F) protein-epithelial cell interactions. Infections by mutant RSV strains deleted of small hydrophobic and/or attachment proteins and only expressing F protein in the envelope were inhibited by prior treatment with CCL5 or a biologically inactive N-terminally modified met-CCL5. Inhibition was also observed when virus adsorption and treatment with CCL5 were performed at 4 degrees C. Flow cytometry further revealed that epithelial cells were positive for CCR3, but not CCR1 or CCR5. Thus, novel mimetics of CCL5 may be useful prophylatic agents to prevent respiratory tract disease caused by RSV.

Immune responses to the nonglycosylated ectodomain of respiratory syncytial virus attachment glycoprotein mediate pulmonary eosinophilia in inbred strains of mice with different MHC haplotypes.

Development of subunit vaccines against respiratory syncytial virus (RSV) for naive human infants is hindered by concerns that immunization with the fusion or attachment (G) proteins will elicit polarized Type 2 T cell responses and cause immunopotentiation upon subsequent natural infection. We investigated the regions of G protein responsible for inducing a Type 2 T cell phenotype in inbred mice of different MHC haplotype toward development of vaccines with improved safety. As demonstrated by IL-5-dependent pulmonary eosinophilia after challenge and serum anti-G protein IgG1 to IgG2 ratios, highly purified native G protein sensitized all strains for a Type 2 T cell phenotype. Stimulation of G protein-primed splenocytes with synthetic overlapping peptides indicated that the nonglycosylated ectodomain was primarily responsible. Respectively the recall responses of BALB/c (H2(d)), C57BL/6 (H-2(b)), SJL (H-2(s)), and C3H/HeJ (H-2(k)) mice were directed against epitopes within peptides spanning amino acids 184-198 (pep(184-198)), 168-181 (pep(168-181)) or 171-185 (pep(171-185)), 176-190 (pep(176-190)), and 104-118 (pep(104-118)) or 159-173 (pep(159-173)). Injection of pep(184-198) conjugated to KLH (pep(184-198)-KLH) primed H2(d) [BALB/c, B6.C-H2(d)/bBy], but not H-2(b) [C57Bl/6, C.B10-H2(b)/LiMcd] mice for pulmonary eosinophilia. Sensitization with a peptide-KLH conjugate encompassing amino acids 149-200 (pep(149-200)-KLH) further confirmed that Type 2 T cell responses in BALB/c, C57BL/6 and SJL, but not C3H/HeJ mice were induced by the nonglycosylated ectodomain of G protein. These data are important for design of safe and efficacious subunit and attenuated vaccines for RSV.