Adaptation of a surface plasmon resonance biosensor with microfluidics for use with small sample volumes and long contact times.

The efficient delivery of sample to surface-immobilized sites is a key element in biosensing. For a surface plasmon resonance (SPR) biosensor, this has been addressed by constant flow through a microfluidic system with a sample injection loop (Sjölander, S.; Urbaniczky, C. Anal. Chem. 1991, 63, 2338-2345). The present study describes an alternative mode of sample delivery without constant unidirectional flow. It was implemented on a commercial Biacore X SPR biosensor equipped with a microfluidic cartridge, but with the fluidic handling performed by an externally computer-controlled syringe pump. We demonstrate that sample volumes as low as 2 microL can be reproducibly positioned to cover the sensor surfaces, manipulated in a serial fashion, efficiently mixed by applying an oscillatory flow pattern, and fully recovered. Compared to the traditional continuous unidirectional flow configuration, we found very similar kinetic responses at high analyte concentrations and slightly slower responses at low concentrations, most likely due to depletion of analyte from the small sample volumes due to surface binding. With the antibody-antigen systems tested, binding parameters were obtained that are generally within 10% of those from conventional experiments. In the new configuration, biosensor experiments can be conducted without the usual constraints in the surface contact time that are correlated with sample volume and mass transport rate. This can translate to improved detection limits for slow reactions and can facilitate kinetic and thermodynamic binding studies.

Systemic or mucosal administration of immunostimulatory DNA inhibits early and late phases of murine allergic conjunctivitis.

Seasonal allergic conjunctivitis is one of the most common manifestations of allergic disease, affecting 15 % population in the United States annually. Short ragweed (RW) is a major cause of seasonal allergies. Immunostimulatory DNA sequences (ISS or CpG motifs) can inhibit an on-going Th2/allergic response and induce a de novo Th1 response. In this study, we investigated the ability of these ISS to modulate allergic responses in a RW-induced mouse model of seasonal allergic conjunctivitis. Systemic or mucosal administration of ISS oligonucleotide (ISS-ODN) after RW sensitization inhibited both the immediate hypersensitivity response and the late-phase cellular infiltration and induced a RW-specific Th1 response. ISS-ODN administration suppressed the rise of RW-specific IgE titers after repeated allergen challenge. Furthermore, ISS administration was more effective than dexamethasone in inhibiting the allergic response. Mechanistically, the ISS-induced immunomodulatory effects were abolished when mice were treated with anti-IL-12 neutralizing antibodies, suggesting a pivotal role for type 1 cytokines in the inhibition of both the immediate hypersensitivity and the late-phase cellular infiltration. Thus, ISS-ODN is a novel anti-inflammatory and immunomodulatory agent that significantly inhibits the allergic response and may provide an alternative to the current standard care of ocular allergy.

The role of IL-12 in the induction of late-phase cellular infiltration in a murine model of allergic conjunctivitis.

BACKGROUND: The applied murine model of allergic conjunctivitis mimics human disease, and an immediate hypersensitivity reaction (IHR) and a late-phase cellular reaction typically develop in sensitized mice after topical challenge with the allergen. OBJECTIVE: We investigated the role of IL-4, IFN-gamma, and IL-12 in the early and late phases of ocular allergy with use of cytokine knockout (KO) mice and neutralizing antibodies. METHODS: Ragweed-sensitized wild-type or IL-4KO, IL-12KO, IFN-gamma KO, anti-IL-12 mAb-treated, recombinant murine IL-12-treated, and anti-IFN-gamma mAb-treated mice were challenged with the allergen 10 days after the immunization. IHR, cellular infiltration, lymphoproliferative response, and cytokine production from draining lymph nodes were recorded and compared among groups. RESULTS: We show that IL-12KO mice and anti-IL-12 antibody-treated wild-type animals failed to have a cellular infiltration into the conjunctiva. Treatment with recombinant murine IL-12 also reduced the number of infiltrating PMNs but increased the percentage of mononuclear cells in the conjunctiva compared with controls. IFN-gamma KO mice had a significantly stronger IHR and prolonged infiltration into the conjunctiva after challenge with ragweed than controls. CONCLUSION: Our data suggest that the presence of IL-12, although better known as a T(H)1-inducing cytokine, is important for the development and the regulation of the late-phase pathologic features in ocular allergy. Furthermore, IFN-gamma is a limiting factor in the late phase of allergy and thus may be important in preventing chronic allergic disease.

Blocking ICAM-1 (CD54) and LFA-1 (CD11a) inhibits experimental allergic conjunctivitis.

Cell adhesion molecules are critical for the homing and migration of leukocytes into inflamed tissues. We investigated the role of ICAM-1 and LFA-1 in a previously described experimental model of ragweed (Rw)-induced allergic conjunctivitis. SWR/J mice were treated intraperitoneally 6 and 1 h prior to topical challenge with Rw with injections of anti-ICAM-1 monoclonal antibody (mAb), anti-LFA-1 mAb, both anti-ICAM-1 and anti-LFA-mAbs, or rat IgG. Blocking ICAM-1 or LFA-1 reduced the clinical signs of allergic conjunctivitis. Treatment with anti-ICAM-1 or anti-LFA-1 mAbs also significantly inhibited cellular infiltration into the conjunctiva. The greatest inhibitory effect was achieved with the combination of antibodies against both cell adhesion molecules. Since antibodies against ICAM-1 and LFA-1 significantly inhibit the development of the clinical and histologic signs of allergic conjunctivitis, they may be useful for treating patients with ocular allergy.

A novel murine model of allergic conjunctivitis.

Allergic conjunctivitis affects over 40 million patients per year in the United States. Here we present the first murine model that incorporates the clinical, cellular, and humoral parameters of allergic conjunctivitis, including a ragweed-induced Th2-type cytokine production by lymphocytes. SWR/J mice were immunized with short ragweed pollen in aluminum hydroxide. Ten days after immunization, allergic conjunctivitis was induced by one topical application of ragweed pollen onto the eye. Immediate response was characterized by chemosis, redness of the conjuctiva, and lid edema. Histopathology and immunohistochemistry showed dense conjunctival infiltration with polymorphonuclear leukocytes, macrophages, and CD4+ T lymphocytes. In addition, ragweed-specific IgG1 and IgE serum levels were significantly higher in immunized animals, and high levels of IL-4 and IL-5 were detected in supernatants from ragweed-activated lymphocytes. This reproducible model is a well-suited instrument for testing the pathophysiology and future therapies of allergic conjunctivitis.

Elevation of laser flare photometry in patients with cytomegalovirus retinitis and AIDS.

PURPOSE: To investigate an alteration of the blood-ocular barriers by laser flare photometry in patients with acquired immunodeficiency syndrome (AIDS) diagnosed with cytomegalovirus retinitis. METHODS: Serial laser flare photometry measurements from 31 eyes of 31 patients with AIDS and newly diagnosed cytomegalovirus retinitis were compared with measurements from 31 control patients with AIDS but without documented eye disease. Location and extent of retinitis, presence of visual symptoms, and CD4 lymphocyte counts were also compared with laser flare photometry readings. RESULTS: Laser flare readings (mean +/- SE) were significantly higher in eyes with (13.0 +/- 1.5 photon counts per msec) than without cytomegalovirus retinitis (4.9 +/- 0.3 photon counts per msec) (P < .001). Lesions within the arcade vessels resulted in significantly higher laser flare photometry readings (17.3 +/- 2.5 photon counts per msec) compared with peripheral retinitis (9.8 +/- 1.5 photon counts per msec) (P = .01). A significant correlation was found between area of involvement of peripheral retinitis and laser flare photometry readings (P = .008). Readings in patients without cytomegalovirus retinitis increased significantly 10 months after the first measurement (9.5 +/- 1.9 photon counts per msec) (P = .04). Readings in patients with cytomegalovirus remained elevated 3 months after successful treatment of retinitis (12.3 +/- 2.3 photon counts per msec) (P = .6). CONCLUSIONS: Laser flare photometry readings are significantly elevated in eyes with cytomegalovirus retinitis, suggesting a breakdown of the blood-ocular barriers. Increasing laser flare photometry readings over time in patients without known ocular disease suggests that HIV infection may cause progressive breakdown of the blood-ocular barrier.