A mechanistically novel peptide agonist of the IL-7 receptor that addresses limitations of IL-7 cytokine therapy.

Interleukin (IL)-7 is broadly active on T-cell populations, and modified versions have been clinically evaluated for a variety of therapeutic applications, including cancer, lymphopenia, and infectious diseases; and found to be relatively well-tolerated and biologically active. Here we describe novel IL-7R agonists that are unrelated in structure to IL-7, bind to the receptor subunits differently from IL-7, but closely emulate IL-7 biology. The small size, low structural complexity, and the natural amino acid composition of the pharmacologically active peptide MDK1472 allows facile incorporation into protein structures, such as the IgG2-Fc fusion MDK-703. This molecule possesses properties potentially better suited to therapeutic applications than native IL-7 or its derivatives. We compared these compounds with IL-7 for immune cell selectivity, induction of IL-7R signaling, receptor-mediated internalization, proliferation, and generation of immune cell phenotypes in human and non-human primate (NHP) peripheral blood cells in vitro; and found them to be similar in biological activity to IL-7. In cynomolgus macaques, MDK-703 exhibits a circulating half-life of 46 hr and produces sustained T-cell expansion characteristic of IL-7 treatment. In the huCD34+-engrafted NSG mouse model of the human immune system, MDK-703 induces an immune cell profile very similar to that generated by IL-7-derived compounds; including the pronounced expansion of memory T-cells, particularly the population of stem-like memory T-cells (Tscm) which may be important for anti-tumor activities reported with IL-7 treatment. Clinical administration of IL-7 and modified variants has been reported to induce anti-drug antibodies (ADAs), including IL-7 neutralizing antibodies. The novel peptide agonist reported here scores very low in predicted immunogenicity, and because the peptide lacks sequence similarity with IL-7, the problematic immunogenic neutralization of endogenous cytokine should not occur. The properties we report here implicate MDK-703 as a candidate for clinical evaluation in oncology, anti-viral and other infectious disease, vaccine enhancement, and treatment of lymphopenia.

Phenotypes of CF rabbits generated by CRISPR/Cas9-mediated disruption of the CFTR gene.

Existing animal models of cystic fibrosis (CF) have provided key insights into CF pathogenesis but have been limited by short lifespans, absence of key phenotypes, and/or high maintenance costs. Here, we report the CRISPR/Cas9-mediated generation of CF rabbits, a model with a relatively long lifespan and affordable maintenance and care costs. CF rabbits supplemented solely with oral osmotic laxative had a median survival of approximately 40 days and died of gastrointestinal disease, but therapeutic regimens directed toward restoring gastrointestinal transit extended median survival to approximately 80 days. Surrogate markers of exocrine pancreas disorders were found in CF rabbits with declining health. CFTR expression patterns in WT rabbit airways mimicked humans, with widespread distribution in nasal respiratory and olfactory epithelia, as well as proximal and distal lower airways. CF rabbits exhibited human CF-like abnormalities in the bioelectric properties of the nasal and tracheal epithelia. No spontaneous respiratory disease was detected in young CF rabbits. However, abnormal phenotypes were observed in surviving 1-year-old CF rabbits as compared with WT littermates, and these were especially evident in the nasal respiratory and olfactory epithelium. The CF rabbit model may serve as a useful tool for understanding gut and lung CF pathogenesis and for the practical development of CF therapeutics.

Inactivation of the miR-183/96/182 Cluster Decreases the Severity of Pseudomonas aeruginosa-Induced Keratitis.

PURPOSE: The microRNA-183/96/182 cluster (miR-183/96/182) plays important roles in sensory organs. Because the cornea is replete with sensory innervation, we hypothesized that miR-183/96/182 modulates the corneal response to bacterial infection through regulation of neuroimmune interactions. METHODS: Eight-week-old miR-183/96/182 knockout (ko) mice and their wild-type littermates (wt) were used. The central cornea of anesthetized mice was scarred and infected with Pseudomonas aeruginosa (PA), strain 19660. Corneal disease was graded at 1, 3, and 5 days postinfection (dpi). Corneal RNA was harvested for quantitative RT-PCR. Polymorphonuclear neutrophils (PMN) were enumerated by myeloperoxidase assays; the number of viable bacteria was determined by plate counts, and ELISA assays were performed to determine cytokine protein levels. A macrophage (Mϕ) cell line and elicited peritoneal PMN were used for in vitro functional assays. RESULTS: MicroRNA-183/96/182 is expressed in the cornea, and in Mϕ and PMN of both mice and humans. Inactivation of miR-183/96/182 resulted in decreased corneal nerve density compared with wt mice. Overexpression of miR-183/96/182 in Mϕ decreased, whereas knockdown or inactivation of miR-183/96/182 in Mϕ and PMN increased their capacity for phagocytosis and intracellular killing of PA. In PA-infected corneas, ko mice showed decreased proinflammatory neuropeptides such as substance P and chemoattractant molecules, MIP-2, MCP1, and ICAM1; decreased number of PMN at 1 and 5 dpi; increased viable bacterial load at 1 dpi, but decreased at 5 dpi; and markedly decreased corneal disease. CONCLUSIONS: MicroRNA-183/96/182 modulates the corneal response to bacterial infection through its regulation of corneal innervation and innate immunity.

Thrombomodulin Protects Against Bacterial Keratitis, Is Anti-Inflammatory, but Not Angiogenic.

PURPOSE: Thrombomodulin (TM) is a multidomain, transmembrane protein with anti-inflammatory properties. Thrombomodulin domain (D) 1 is lectin-like, interacting with Lewis Y antigen on lipopolysaccharide, and with HMGB1, while TMD23 is associated with angiogenic and anti-inflammatory functions. Thus, we tested if TM is protective against Pseudomonas aeruginosa keratitis and whether it enhanced corneal vascularity. METHODS: Eyes of C57BL/6 (B6) mice were injected with recombinant TM (rTM), rTMD1, or PBS subconjunctivally before and intraperitoneally after infection with P. aeruginosa. Clinical scores, photography with a slit lamp, RT-PCR, ELISA, myeloperoxidase (MPO) assay, viable bacterial plate counts, and India ink perfusion were used to assess the disease response and corneal vascularity (rTM only). RESULTS: Recombinant TM versus PBS treatment reduced clinical scores and corneal opacity. Corneal mRNA levels for HMGB1 were unchanged, but proinflammatory molecules IL-1ß, CXCL2, NF-κB, TLR4, and RAGE were decreased; anti-inflammatory molecules SIGIRR and ST2 were increased. ELISA confirmed the mRNA data for HMGB1, IL-1ß, and CXCL2 proteins. Both neutrophil influx and viable bacterial plate counts also were decreased after rTM treatment. Protein levels for angiogenic molecules VEGF, VEGFR-1, and VEGFR-2 were measured at 5 days post infection and were not different or reduced significantly after rTM treatment. Further, perfusion with India ink revealed similar vessel ingrowth between the two groups. Similar studies were performed with rTMD1, but disease severity, mRNA, proteins, MPO, and plate counts were not changed from controls. CONCLUSIONS: These data provide evidence that rTM treatment is protective against bacterial keratitis, does not reduce HMGB1, and is not angiogenic.

Interleukin 17 regulates Mer tyrosine kinase-positive cells in Pseudomonas aeruginosa keratitis.

PURPOSE: To determine if IL-17 regulates Mer tyrosine kinase-positive (MerTK+) cells in Pseudomonas aeruginosa keratitis. METHODS: Interleukin 17 was tested in normal and infected cornea of susceptible C57BL/6 and resistant BALB/c mice. The latter were treated with recombinant mouse (rm) IL-17; both groups were treated with IL-17 neutralizing antibody. Mice were infected, and clinical score, PCR, ELISA, and myeloperoxidase (MPO) assays tested expression of proinflammatory and anti-inflammatory mediators and polymorphonuclear neutrophilic leukocyte (PMN) infiltrate. Fas and Fas ligand (FasL) protein levels were assessed in both mouse strains, while MerTK+ cells were examined by immunostaining and cell sorting before and after IL-17 neutralization. RESULTS: The IL-17 mRNA and protein were higher in C57BL/6 versus BALB/c cornea after infection. The rmIL-17 treatment of BALB/c mice modified proinflammatory and anti-inflammatory mediators, but clinical score and MPO assay revealed no differences. However, only BALB/c mice treated with IL-17 neutralizing antibody showed increased disease, macrophage inflammatory protein (MIP) 2, and MPO levels. Fas and FasL protein levels, elevated earlier in BALB/c versus C57BL/6 mice, correlated with significantly more MerTK+ cells in BALB/c cornea at 3 days after infection. Neutralization of IL-17 in C57BL/6 mice elevated MerTK+ cells, while similar treatment of BALB/c mice significantly decreased them. CONCLUSIONS: These data provide evidence that IL-17 expression is higher in C57BL/6 versus BALB/c cornea after infection and that the latter group has more MerTK+ cells. Exogenous rmIL-17 failed to shift the disease response in resistant mice, but its neutralization resulted in worsened disease and reduced MerTK+ cells. Neutralization of IL-17 in C57BL/6 mice increased MerTK+ cells but did not dramatically shift the disease response.

Effects of VIP on corneal reconstitution and homeostasis following Pseudomonas aeruginosa induced keratitis.

PURPOSE: Studies from our laboratory have demonstrated that vasoactive intestinal peptide (VIP) directly converts the normally susceptible C57BL/6J (B6) mouse to resistant after ocular infection through modulation of the inflammatory response. This study examines mechanisms by which VIP influences the healing phase following infection--specifically reconstitution of the extracellular matrix (ECM). METHODS: B6 mice received daily intraperitoneal (IP) injections of VIP, while control mice were similarly injected with sterile phosphate buffered saline (PBS). Real-time RT-PCR, ELISA, and immunofluorescent staining were used to assess the effects of VIP treatment on ECM molecule expression after Pseudomonas aeruginosa-induced keratitis. We also compared the effect of VIP treatment on lipopolysaccharide (LPS)-stimulated B6- and BALB/c-derived fibroblasts. RESULTS: In vivo analyses revealed that VIP treatment of P. aeruginosa-infected B6 corneas led to a significant increase in ECM molecules associated with healing/homeostasis, while those associated with ECM degradation were significantly down-regulated when compared to wild-type (WT) controls. In vitro studies revealed that VIP treatment of lipopolysaccharide-stimulated fibroblasts derived from susceptible B6 and resistant BALB/c mice expressed distinct differences in ECM molecule expression, whereby the latter expressed higher levels of ECM molecules aimed at reconstitution. Furthermore, differential expression of VIP receptor-1/VIP receptor-2 (VIPR1/VIPR2) was observed between B6 and BALB/c after VIP treatment of LPS-stimulated fibroblasts. CONCLUSIONS: VIP treatment functions to enhance ECM reconstitution, which appears to be carried out in large part by fibroblasts via VIPR2. Overall, the data from this study suggest that VIP not only regulates disease pathogenesis, but also functions to restore integrity of the corneal stroma.

The role of VIP in cornea.

PURPOSE: Exogenous vasoactive intestinal peptide (VIP) down-regulates pro-inflammatory but up-regulates anti-inflammatory cytokines, growth factors (GFs) and Toll-like receptors promoting healing in experimental Pseudomonas aeruginosa (P. aeruginosa) keratitis. Whether VIP is required for GF or GF receptor (R) expression in normal and infected corneas is unknown and is the purpose of this study. METHODS: VIP knockout ((-/-)) and wild-type (WT) C57BL/6 (B6) mice were infected and tested using PCR array, real-time RT-PCR, ELISA, and immunostaining. VIP antagonist treatment studies also were done using B6 and BALB/c mice. RESULTS: Infected corneas of VIP(-/-) versus WT B6 mice perforated earlier (2 vs. 5 days postinfection [p.i.]), and array data showed that GFs were differentially changed between groups. RT-PCR revealed that the infected cornea of VIP(-/-) versus WT mice expressed higher mRNA levels of epidermal growth factor (EGF) and hepatocyte growth factor (HGF), reduced FGF, EGFR, and HGFR, with no difference in FGFR; differences between groups were not seen in normal cornea. Immunostaining for GF and GFR in the normal cornea of VIP(-/-) versus WT mice was similar. However, at 1 day p.i., VIP(-/-) versus WT mice had more intense EGF and HGF, similar FGFR, and reduced FGF, EGFR, and HGFR staining. VIP antagonist treatment decreased protein levels for GFR at 5 days p.i. in both B6 and BALB/c mice, with no significant changes in normal cornea. CONCLUSIONS: The data showed that endogenous VIP is not requisite for GF or GFR expression in the normal cornea but, after infection, its absence or reduction is critical for their regulation.

Substance P affects growth factors in Pseudomonas aeruginosa-infected mouse cornea.

PURPOSE: This study analyzed the influence of substance P (SP) on growth factors related to wound healing in mice in the presence of infectious keratitis. METHODS: Naturally resistant mice were injected intraperitoneally with SP or phosphate-buffered saline and infected with Pseudomonas aeruginosa, and corneal messenger RNA (mRNA) levels of growth factors and apoptosis genes were tested. Enzyme-linked immunosorbent assay determined the protein levels, whereas immunohistochemistry tested the distribution, macrophage phenotype, and cell quantitation. In vitro, macrophages were stimulated with lipopolysaccharide (LPS; with or without SP) and mRNA levels of proinflammatory and antiinflammatory cytokines and apoptosis genes were tested. RESULTS: After SP, epidermal growth factor mRNA and protein levels were disparately regulated early, with no differences later in the disease. Hepatocyte growth factor and fibroblast growth factor-7 mRNA and protein levels were increased after SP treatment. Enumerating dual-labeled stromal cells revealed no difference between SP-treated versus phosphate-buffered saline-treated groups in the percentage of epidermal growth factor-labeled fibroblasts or macrophages, but there were significant increases in both hepatocyte growth factor- and fibroblast growth factor-7-labeled cells. Type 2 (M2) macrophages and caspase-3 mRNA levels were decreased, whereas B-cell lymphoma-2 mRNA expression was increased after SP treatment. In vitro, mRNA levels of several proinflammatory cytokines and B-cell lymphoma-2 were elevated, whereas transforming growth factor ß was decreased after macrophage stimulation with SP (with LPS) over LPS alone. (Mice: n = 105 control; 105 experimental.) CONCLUSIONS: These data show that treatment with SP in infectious keratitis elevates growth factors but also adversely affects the disease by enhancing the inflammatory response and its sequelae.

Vasoactive intestinal peptide downregulates proinflammatory TLRs while upregulating anti-inflammatory TLRs in the infected cornea.

TLRs recognize microbial pathogens and trigger an immune response, but their regulation by neuropeptides, such as vasoactive intestinal peptide (VIP), during Pseudomonas aeruginosa corneal infection remains unexplored. Therefore, C57BL/6 (B6) mice were injected i.p. with VIP, and mRNA, protein, and immunostaining assays were performed. After VIP treatment, PCR array and real-time RT-PCR demonstrated that proinflammatory TLRs (conserved helix-loop-helix ubiquitous kinase, IRAK1, TLR1, TLR4, TLR6, TLR8, TLR9, and TNFR-associated factor 6) were downregulated, whereas anti-inflammatory TLRs (single Ig IL-1-related receptor [SIGIRR] and ST2) were upregulated. ELISA showed that VIP modestly downregulated phosphorylated inhibitor of NF-κB kinase subunit α but upregulated ST2 ~2-fold. SIGIRR was also upregulated, whereas TLR4 immunostaining was reduced in cornea; all confirmed the mRNA data. To determine whether VIP effects were cAMP dependent, mice were injected with small interfering RNA for type 7 adenylate cyclase (AC7), with or without VIP treatment. After silencing AC7, changes in mRNA levels of TLR1, TNFR-associated factor 6, and ST2 were seen and unchanged with addition of VIP, indicating that their regulation was cAMP dependent. In contrast, changes were seen in mRNA levels of conserved helix-loop-helix ubiquitous kinase, IRAK1, 2, TLR4, 9 and SIGIRR following AC7 silencing alone; these were modified by VIP addition, indicating their cAMP independence. In vitro studies assessed the effects of VIP on TLR regulation in macrophages and Langerhans cells. VIP downregulated mRNA expression of proinflammatory TLRs while upregulating anti-inflammatory TLRs in both cell types. Collectively, the data provide evidence that VIP downregulates proinflammatory TLRs and upregulates anti-inflammatory TLRs and that this regulation is both cAMP dependent and independent and involves immune cell types found in the infected cornea.

VIP and growth factors in the infected cornea.

PURPOSE: Vasoactive intestinal peptide (VIP) is an anti-inflammatory neuropeptide that downregulates proinflammatory cytokines and promotes healing in a susceptible model of P. aeruginosa keratitis. Growth factors also play a role in corneal healing and restoration of tissue homeostasis after wounding. However, whether VIP treatment modulates growth factors to promote healing in the infected cornea remains untested and is the purpose of this study. METHODS: C57BL/6 (B6) mice were injected with VIP and mRNA and protein levels, and immunostaining for EGF, FGF, HGF, and VEGF-A were done. Exogenous treatment with a mixture of the growth factors also was tested and levels of cytokines, defensins, and bacterial counts were determined. RESULTS: Real-time RT-PCR, immunostaining, and ELISA data demonstrated that treatment with VIP enhanced levels of EGF, FGF, and HGF during disease, and that VEGF-A, and associated angiogenic molecules also were increased by VIP. Moreover, immunohistochemical studies confirmed that both epithelial and stromal cells participated in growth factor production. Most notably, treatment with a mixture of EGF, FGF, and HGF after disease onset, prevented corneal perforation when compared with controls. This outcome was associated with downregulation of proinflammatory cytokines such as macrophage inflammatory protein-2 (MIP-2), upregulation of anti-inflammatory cytokines such as TGF-ß, and antimicrobials ß-defensins 2 and 3, as well as decreased plate counts at 1 day postinfection (p.i.) (P = 0.0001). CONCLUSIONS: Collectively, the data provide evidence that VIP treatment modulates growth factors, angiogenic molecules, and defensins in the infected cornea and that this in turn promotes healing and restoration of tissue homeostasis.

VIP promotes resistance in the Pseudomonas aeruginosa-infected cornea by modulating adhesion molecule expression.

PURPOSE: This study tested the hypothesis that the neuropeptide vasoactive intestinal peptide (VIP) regulates adhesion molecule expression, reduces inflammatory cell migration and infiltration into the Pseudomonas aeruginosa-infected cornea of susceptible B6 mice, and promotes corneal healing and resistance. METHODS: B6 mice received daily intraperitoneal (IP) injections of VIP from -1 through 5 days after infection. Control mice were similarly injected with sterile phosphate-buffered saline (PBS). Transcript levels of adhesion molecules were determined by PCR array, then select molecules were tested individually by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and confirmed at the protein level by enzyme-linked immunosorbent assay (ELISA) or immunofluorescent staining with confocal laser scanning microscopy at various time points after infection to assess the effects of VIP treatment in the regulation of adhesion molecule expression. RESULTS: Injection of B6 mice with VIP compared with PBS resulted in significant downregulation of intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, platelet-endothelial cell adhesion molecule-1, and P-selectin and L-selectin mRNA expression. Protein levels for ICAM-1 and VCAM-1, detected by ELISA, supported the mRNA data at similar time points. Immunofluorescence staining further confirmed the effects of VIP treatment, showing reduced corneal expression of ICAM-1/leukocyte function-associated antigen (LFA-1) and VCAM-1/very late antigen-4 (VLA-4) at select time points compared with PBS-treated animals. CONCLUSIONS: VIP treatment downregulates the production of adhesion molecules integral to the transmigration process of host inflammatory cells (polymorphonuclear neutrophils, macrophages) into the infected cornea. This results directly in reduced cellular infiltration, less stromal destruction, and better disease outcome.

Role of the Fas pathway in Pseudomonas aeruginosa keratitis.

PURPOSE: The role of the Fas pathway was tested in Pseudomonas aeruginosa-infected mouse cornea by contrasting the responses of FasL(-/-) and wild-type (WT) mice. METHODS: TUNEL staining, real-time RT-PCR, immunostaining, and ELISA assay were used. RESULTS: Compared with WT (resistant) mice, BALB/c FasL(-/-) exhibited significantly elevated bacterial counts and polymorphonuclear leukocyte numbers at 1 and 3 days postinfection (p.i.) and worse outcomes from disease. Similar bacterial challenges in C57BL/6 FasL(-/-) compared with WT mice also led to worsened disease as evidenced by earlier corneal perforation in the susceptible mouse strain. Intense TUNEL staining of apoptotic cells was seen earlier (1 day vs. 3 days) p.i. in BALB/c WT than in knockout mice, This earlier apoptotic pattern correlated with increased expression of caspases 3, 8, and 9 and BAX and with decreased expression of the antiapoptotic molecule Bcl-2. Furthermore, expression levels of the proinflammatory molecule TNF-alpha and its receptor, MIP-2, inducible nitric oxide synthase (iNOS), and nitrite also were significantly elevated in the infected cornea of BALB/c FasL(-/-) compared with WT mice. In vitro, LPS-stimulated Mphi from BALB/c FasL(-/-) mice expressed significantly less caspase 3 and 9, BAX, and IL-10 and more TNF-alpha, MIP-2, and IL-1beta than did cells from WT mice. CONCLUSIONS: Fas-FasL interaction in the cornea balances the host innate immune response to improve disease outcome by promoting earlier apoptosis and regulating proinflammatory cytokines/chemokines and nitric oxide (nitrite) production. Dysregulation of this interaction contributes to bystander tissue damage, enhancing nutrients for bacterial growth and worsened disease outcome after P. aeruginosa infection.

Rat silicone hydrogel contact lens model: effects of high- versus low-Dk lens wear.

OBJECTIVES: This study used a rat contact lens (CL) model to test if high- versus low-Dk lens wear caused changes in (1) conjunctival Langerhans cell (LC) number or location; (2) Bcl-2 expression; and (3) infection risk. METHODS: Female, Lewis rats wore a high- or low-Dk CL continuously for 2 weeks. Afterward, corneas were harvested and processed for ADPase activity to identify LCs, for immunostaining and for real time-polymerase chain reaction. Contact lens-wearing rats also were challenged with Pseudomonas aeruginosa by placing a bacterial-soaked CL on the eye followed by topical delivery of bacteria. After 48 hrs, slit lamp examination and real time-polymerase chain reaction were used to evaluate the corneal response. RESULTS: Conjunctival LC were significantly increased after low- versus high-Dk CL wear (P<0.0001). In contrast, conjunctival LC in non-lens wearing rats was not significantly different from the high-Dk lens wearing group. Bcl-2 mRNA levels were significantly decreased in low- versus high-Dk CL wearing rats, while Bax, FasL, caspase 3, and caspase 9 levels were unchanged. Immunostaining for Bcl-2 showed fewer positively stained epithelial cells in the low- versus high-Dk lens wearing group. After bacterial challenge, 30% of low- versus none of the high-Dk CL wearing corneas became infected and showed increased mRNA levels for several proinflammatory cytokines/chemokines, inducible nitric oxide synthase and matrix metalloproteinase-9. CONCLUSION: Low- versus high-Dk or non-CL wear led to an increased number of conjunctival LC, decreased Bcl-2 levels, and increased the risk of bacterial infection.

Substance P promotes susceptibility to Pseudomonas aeruginosa keratitis in resistant mice: anti-inflammatory mediators downregulated.

PURPOSE: Studies have shown that blocking substance P (SP) binding to neurokinin 1 receptor with spantide I prevents Pseudomonas aeruginosa-induced corneal perforation in susceptible C57BL/6 mice. This study tested the effect of SP injection on the resistance response (cornea heals) of BALB/c mice. METHODS: The day before infection, mice were injected intraperitoneally with SP or PBS. Disease was graded by clinical score, slit lamp, plate count, real-time RT-PCR, and ELISA assays, and polymorphonuclear neutrophils (PMNs) were quantitated using a myeloperoxidase assay. In additional experiments, BALB/c mice were injected intraperitoneally with vasoactive intestinal peptide (VIP) antagonist and similarly analyzed. RESULTS: Mice injected with SP exhibited worsened disease on days 1 to 7 after infection compared with controls. SP injection resulted in elevated PMN levels and viable bacterial counts in the cornea 3 and 5 days after infection. mRNA expression for NFkappaB and type 1 cytokines (e.g., IFN-gamma), as well as for TNF-alpha, MIP-2, IL-18, IL-6, and IL-1beta, were significantly elevated, whereas VIP and cytokines TGF-beta and IL-10 were significantly reduced. Differences in mRNA expression were selectively confirmed at the protein level by ELISA for NFkappaB, IL-1beta, and IL-10. VIP antagonist treatment also resulted in exacerbated disease scores, elevated proinflammatory mediators, and reduced anti-inflammatory mediators. CONCLUSIONS: These data provide evidence that the neuropeptide SP, among its broad systemic effects, is a potent neuroimmunoregulator that promotes susceptibility in the resistant BALB/c mouse by overcoming the anti-inflammatory effects of VIP and IL-10 and that a balance between SP and VIP levels may be critical in disease resolution.

Constitutive excitation by Gly90Asp rhodopsin rescues rods from degeneration caused by elevated production of cGMP in the dark.

Previous experiments indicate that congenital human retinal degeneration caused by genetic mutations that change the Ca(2+) sensitivity of retinal guanylyl cyclase (retGC) can result from an increase in concentration of free intracellular cGMP and Ca(2+) in the photoreceptors. To rescue degeneration in transgenic mouse models having either the Y99C or E155G mutations of the retGC modulator guanylyl cyclase-activating protein 1 (GCAP-1), which produce elevated cGMP synthesis in the dark, we used the G90D rhodopsin mutation, which produces constitutive stimulation of cGMP hydrolysis. The effects of the G90D transgene were evaluated by measuring retGC activity biochemically, by recording single rod and electroretinogram (ERG) responses, by intracellular free Ca(2+) measurement, and by retinal morphological analysis. Although the G90D rhodopsin did not alter the abnormal Ca(2+) sensitivity of retGC in the double-mutant animals, the intracellular free cGMP and Ca(2+) concentrations returned close to normal levels, consistent with constitutive activation of the phosphodiesterase PDE6 cascade in darkness. G90D decreased the light sensitivity of rods but spared them from severe retinal degeneration in Y99C and E155G GCAP-1 mice. More than half of the photoreceptors remained alive, appeared morphologically normal, and produced electrical responses, at the time when their siblings lacking the G90D rhodopsin transgene lost the entire retinal outer nuclear layer and no longer responded to illumination. These experiments indicate that mutations that lead to increases in cGMP and Ca(2+) can trigger photoreceptor degeneration but that constitutive activation of the transduction cascade in these animals can greatly enhance cell survival.

Spantide I decreases type I cytokines, enhances IL-10, and reduces corneal perforation in susceptible mice after Pseudomonas aeruginosa infection.

PURPOSE: To determine the effects of blocking substance P (SP) interactions with its major receptor (NK1-R) using the antagonist spantide I in susceptible mice infected with Pseudomonas aeruginosa. METHODS: Immunohistochemistry and enzyme immunosorbent assay (EIA) tested levels of SP in the cornea of B6 and BALB/c mice. B6 mice were treated with spantide, and after infection, slit lamp examination; clinical score; bacterial counts; and myeloperoxidase (MPO), RT-PCR, ELISA, and polymorphonuclear (PMN) cell chemotaxis assays were performed. RESULTS: SP corneal levels were significantly elevated constitutively and after infection in the B6 more than in BALB/c mice. Spantide treatment of B6 mice significantly decreased the number of perforated corneas, bacterial counts, and PMNs. mRNA levels for type I cytokines (e.g., IFN-gamma) as well as MIP-2, IL-6, TNF-alpha, and IL-1beta (mRNA and protein) also were significantly reduced after spantide treatment. The type II cytokine IL-10 (mRNA and protein) was elevated, whereas TGF-beta mRNA levels were unchanged after spantide treatment. PMN chemotaxis was induced by SP and other neuropeptides in vitro, but was not affected by spantide I. mRNA for neurokinin-1-receptor-1 (NK-1R) was detected in the normal and infected corneas and on macrophages (Mphis), but not on PMNs (unstimulated or stimulated with endotoxin [LPS]). Spantide treatment of Mphis reduced IL-1beta after LPS+SP treatment but not after either alone. CONCLUSIONS: The SP antagonist Spantide provides a novel approach to reduce type 1 and enhance the type 2 cytokine IL-10 in the infected cornea of B6 mice, leading to a significant reduction in corneal perforation and improved disease outcome.

Vasoactive intestinal peptide balances pro- and anti-inflammatory cytokines in the Pseudomonas aeruginosa-infected cornea and protects against corneal perforation.

Corneal infection with Pseudomonas aeruginosa perforates the cornea in susceptible C57BL/6 (B6), but not resistant BALB/c, mice. To determine whether vasoactive intestinal peptide (VIP) played a role in development of the resistant response, protein expression levels were tested by immunocytochemistry and enzyme immunoassay in BALB/c and B6 corneas. Both mouse strains showed constitutive expression of corneal VIP protein and nerve fiber distribution. However, disparate expression patterns were detected in the cornea after infection. VIP protein was elevated significantly in BALB/c over B6 mice at 5 and 7 days postinfection. Therefore, B6 mice were injected with rVIP and subsequently demonstrated decreased corneal opacity and resistance to corneal perforation compared with PBS controls. rVIP- vs PBS-treated B6 mice also demonstrated down-regulation of corneal mRNA and/or protein levels for proinflammatory cytokines/chemokines: IFN-gamma, IL-1beta, MIP-2, and TNF-alpha, whereas anti-inflammatory mediators, IL-10 and TGF-beta1, were up-regulated. Treatment with rVIP decreased NO levels and polymorphonuclear neutrophil (PMN) number. To further define the role of VIP, peritoneal macrophages (Mphi) and PMN from BALB/c and B6 mice were stimulated with LPS and treated with rVIP. Treatment of LPS-stimulated Mphi from both mouse strains resulted in decreased IL-1beta and MIP-2 protein levels; PMN responded similarly. Both cell types also displayed a strain-dependent differential response to rVIP, whereby B6 Mphi/PMN responded only to a higher concentration of VIP compared with cells from BALB/c mice. These data provide evidence that neuroimmune regulation of the cytokine network and host inflammatory cells functions to promote resistance against P. aeruginosa corneal infection.

Religion and spirituality in coping with breast cancer: perspectives of Chilean women.

This study examined the roles of religion and spirituality in relation to coping with breast cancer in Chilean women. Specifically, the purpose of the study was to examine how these patients used religion and spirituality to cope with their illness; how their illness changed the roles of religion and spirituality in their lives; and their views regarding whether, and if so how, spiritual faith can help patients recuperate from breast cancer. Twenty-seven women with breast cancer who were patients at a clinic in Santiago, Chile were recruited to participate in one-on-one interviews. The transcribed interviews were analyzed using the "constant comparative method" to seek patterns and organize the content into specific themes. Women viewed religion and spirituality as primary resources for themselves and others to use in coping with breast cancer. Women's use of religion and spirituality was manifested in praying, in their perceived dependence on God to intercede and guide them through their illness, and in obtaining social support from other persons in their faith community. Half (13/26) of the women reported that their cancer prompted an increased emphasis on religion and spirituality in their lives by deepening their faith in God. Almost all (26/27) participants endorsed the belief that spiritual faith can help cancer patients to recuperate. These findings suggest that health care providers working should be aware of the culturally dependent roles that religion and spirituality play in women's coping with breast cancer.

Allosteric activation of the follicle-stimulating hormone (FSH) receptor by selective, nonpeptide agonists.

The pituitary glycoprotein hormones, luteinizing hormone and follicle-stimulating hormone (FSH), act through their cognate receptors to initiate a series of coordinated physiological events that results in germ cell maturation. Given the importance of FSH in regulating folliculogenesis and fertility, the development of FSH mimetics has been sought to treat infertility. Currently, purified and recombinant human FSH are the only FSH receptor (FSH-R) agonists available for infertility treatment. By screening unbiased combinatorial chemistry libraries, using a cAMP-responsive luciferase reporter assay, we discovered thiazolidinone agonists (EC50's = 20 microm) of the human FSH-R. Subsequent analog library screening and parallel synthesis optimization resulted in the identification of a potent agonist (EC50 = 2 nm) with full efficacy compared with FSH that was FSH-R-selective and -dependent. The compound mediated progesterone production in Y1 cells transfected with the human FSH-R (EC50 = 980 nm) and estradiol production from primary rat ovarian granulosa cells (EC50 = 10.5 nm). This and related compounds did not compete with FSH for binding to the FSH-R. Use of human FSH/thyroid-stimulating hormone (TSH) receptor chimeras suggested a novel mechanism for receptor activation through a binding site independent of the natural hormone binding site. This study is the first report of a high affinity small molecule agonist that activates a glycoprotein hormone receptor through an allosteric mechanism. The small molecule FSH receptor agonists described here could lead to an oral alternative to the current parenteral FSH treatments used clinically to induce ovarian stimulation for both in vivo and in vitro fertilization therapy.

Pseudomonas aeruginosa-induced inflammation in the rat extended-wear contact lens model.

PURPOSE: To examine the early host response to Pseudomonas aeruginosa challenge in the extended contact lens-wearing rat model. METHODS: Lewis rats were fitted with extended-wear lotrafilcon A hydrogel lenses in the left eye, and the right eye served as the control. Bacterial challenge was initiated in the experimental eye by fitting a bacteria-soaked contact lens and by topical delivery of the bacteria. On first detection of corneal opacity, slitlamp examination, histopathologic examination, viable bacteria counts, enzyme-linked immunosorbent assay, myeloperoxidase, Langerhans cell detection, and multiprobe ribonuclease protection assays were used to evaluate the early corneal response. RESULTS: Analysis of bacterially challenged contact lens-wearing versus control rats showed Langerhans cells and polymorphonuclear neutrophils only in the experimentally challenged cornea. In addition, in the experimentally challenged cornea, ribonuclease protection and enzyme-linked immunosorbent analyses showed an upregulation of proinflammatory cytokines, including interleukins 1beta and 6, suggesting that with contact lens wear, these cytokines contribute to the early corneal response and, potentially, disease. CONCLUSIONS: The contact lens-wearing rat model allows a unique analysis of the early effects of bacterial challenge in extended-wear contact lenses in the absence of corneal scarring, used in most rodent models. The rat model should be valuable to delineate further the effects of contact lens wear, including the testing of additional contact lens-related complications.